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1.
Hum Mol Genet ; 31(5): 665-673, 2022 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-34508592

RESUMO

Autosomal dominant lateral temporal epilepsy (ADLTE) is a genetically heterogeneous neurologic disorder clinically characterized by focal seizures with auditory symptoms and/or aphasia. About 20% of ADLTE families segregate disease-causing heterozygous mutations in RELN, a brain-expressed gene encoding the secreted protein Reelin. Using a cell-based secretion assay, we show that pathogenic RELN mutations abolish or significantly reduce secretion of mutant proteins and that this secretion defect results from impaired trafficking of mutant Reelin along the secretory pathway. Confocal immunofluorescence analysis of transiently transfected cells shows that Reelin mutant proteins are degraded by the autophagy system, as revealed by increased formation of autophagosomes immunoreacting with the autophagy markers p62 and LC3. In addition, LC3 immunoblotting shows a significant increase of autophagy flux due to mutant overexpression. Finally, we show that the secretion defect of mutant proteins can be partially rescued by small-molecule correctors. Altogether, these results suggest that Reelin mutant proteins are not properly secreted and that they are degraded through the autophagy pathway.


Assuntos
Epilepsia do Lobo Temporal , Proteínas do Tecido Nervoso , Epilepsia do Lobo Temporal/genética , Humanos , Proteínas Mutantes/genética , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteína Reelina , Serina Proteases/genética
2.
Ann Neurol ; 94(5): 825-835, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37597255

RESUMO

OBJECTIVE: Familial mesial temporal lobe epilepsy (FMTLE) is an important focal epilepsy syndrome; its molecular genetic basis is unknown. Clinical descriptions of FMTLE vary between a mild syndrome with prominent déjà vu to a more severe phenotype with febrile seizures and hippocampal sclerosis. We aimed to refine the phenotype of FMTLE by analyzing a large cohort of patients and asked whether common risk variants for focal epilepsy and/or febrile seizures, measured by polygenic risk scores (PRS), are enriched in individuals with FMTLE. METHODS: We studied 134 families with ≥ 2 first or second-degree relatives with temporal lobe epilepsy, with clear mesial ictal semiology required in at least one individual. PRS were calculated for 227 FMTLE cases, 124 unaffected relatives, and 16,077 population controls. RESULTS: The age of patients with FMTLE onset ranged from 2.5 to 70 years (median = 18, interquartile range = 13-28 years). The most common focal seizure symptom was déjà vu (62% of cases), followed by epigastric rising sensation (34%), and fear or anxiety (22%). The clinical spectrum included rare cases with drug-resistance and/or hippocampal sclerosis. FMTLE cases had a higher mean focal epilepsy PRS than population controls (odds ratio = 1.24, 95% confidence interval = 1.06, 1.46, p = 0.007); in contrast, no enrichment for the febrile seizure PRS was observed. INTERPRETATION: FMTLE is a generally mild drug-responsive syndrome with déjà vu being the commonest symptom. In contrast to dominant monogenic focal epilepsy syndromes, our molecular data support a polygenic basis for FMTLE. Furthermore, the PRS data suggest that sub-genome-wide significant focal epilepsy genome-wide association study single nucleotide polymorphisms are important risk variants for FMTLE. ANN NEUROL 2023;94:825-835.


Assuntos
Epilepsia do Lobo Temporal , Convulsões Febris , Humanos , Pré-Escolar , Criança , Adolescente , Adulto Jovem , Adulto , Pessoa de Meia-Idade , Idoso , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/diagnóstico , Estudo de Associação Genômica Ampla , Convulsões Febris/genética , Imageamento por Ressonância Magnética , Eletroencefalografia , Síndrome , Hipocampo
3.
Ann Neurol ; 83(3): 483-493, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29394500

RESUMO

OBJECTIVE: Autosomal-dominant lateral temporal epilepsy (ADLTE) is a genetic focal epilepsy characterized by auditory symptoms. Two genes, LGI1 and RELN, encoding secreted proteins, are implicated in the etiology of ADLTE, but half of the affected families remain genetically unsolved, and the underlying molecular mechanisms are yet to be clarified. We aimed to identify additional genes causing ADLTE to better understand the genetic basis and molecular pathway underlying this epileptic disorder. METHODS: A cohort of Italian ADLTE families was examined by whole exome sequencing combined with genome-wide single-nucleotide polymorphism-array linkage analysis. RESULTS: We identified two ADLTE-causing variants in the MICAL-1 gene: a p.Gly150Ser substitution occurring in the enzymatically active monooxygenase (MO) domain and a p.Ala1065fs frameshift indel in the C-terminal domain, which inhibits the oxidoreductase activity of the MO domain. Each variant segregated with ADLTE in a single family. Examination of candidate variants in additional genes excluded their implication in ADLTE. In cell-based assays, both variants significantly increased MICAL-1 oxidoreductase activity and induced cell contraction in COS7 cells, which likely resulted from deregulation of F-actin dynamics. INTERPRETATION: MICAL-1 oxidoreductase activity induces disassembly of actin filaments, thereby regulating the organization of the actin cytoskeleton in developing and adult neurons and in other cell types. This suggests that dysregulation of the actin cytoskeleton dynamics is a likely mechanism by which MICAL-1 pathogenic variants lead to ADLTE. Ann Neurol 2018;83:483-493.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas do Citoesqueleto/genética , Epilepsia do Lobo Temporal/diagnóstico , Epilepsia do Lobo Temporal/genética , Variação Genética/genética , Proteínas com Domínio LIM/genética , Mutação/genética , Adulto , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Estudos de Coortes , Feminino , Humanos , Itália , Masculino , Proteínas dos Microfilamentos , Pessoa de Meia-Idade , Oxigenases de Função Mista , Linhagem , Proteína Reelina , Adulto Jovem
4.
PLoS Genet ; 12(10): e1006376, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27760137

RESUMO

Autosomal dominant lateral temporal epilepsy (ADTLE) is a focal epilepsy syndrome caused by mutations in the LGI1 gene, which encodes a secreted protein. Most ADLTE-causing mutations inhibit LGI1 protein secretion, and only a few secretion-positive missense mutations have been reported. Here we describe the effects of four disease-causing nonsynonymous LGI1 mutations, T380A, R407C, S473L, and R474Q, on protein secretion and extracellular interactions. Expression of LGI1 mutant proteins in cultured cells shows that these mutations do not inhibit protein secretion. This finding likely results from the lack of effects of these mutations on LGI1 protein folding, as suggested by 3D protein modelling. In addition, immunofluorescence and co-immunoprecipitation experiments reveal that all four mutations significantly impair interaction of LGI1 with the ADAM22 and ADAM23 receptors on the cell surface. These results support the existence of a second mechanism, alternative to inhibition of protein secretion, by which ADLTE-causing LGI1 mutations exert their loss-of-function effect extracellularly, and suggest that interactions of LGI1 with both ADAM22 and ADAM23 play an important role in the molecular mechanisms leading to ADLTE.


Assuntos
Proteínas ADAM/genética , Epilepsia do Lobo Frontal/genética , Proteínas do Tecido Nervoso/genética , Mapas de Interação de Proteínas/genética , Proteínas/genética , Transtornos do Sono-Vigília/genética , Proteínas ADAM/química , Proteínas ADAM/metabolismo , Substituição de Aminoácidos/genética , Animais , Células COS , Membrana Celular/genética , Membrana Celular/metabolismo , Chlorocebus aethiops , Epilepsia do Lobo Frontal/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Conformação Proteica , Dobramento de Proteína , Proteínas/química , Proteínas/metabolismo , Transtornos do Sono-Vigília/patologia
5.
Am J Hum Genet ; 96(6): 992-1000, 2015 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-26046367

RESUMO

Autosomal-dominant lateral temporal epilepsy (ADLTE) is a genetic epilepsy syndrome clinically characterized by focal seizures with prominent auditory symptoms. ADLTE is genetically heterogeneous, and mutations in LGI1 account for fewer than 50% of affected families. Here, we report the identification of causal mutations in reelin (RELN) in seven ADLTE-affected families without LGI1 mutations. We initially investigated 13 ADLTE-affected families by performing SNP-array linkage analysis and whole-exome sequencing and identified three heterozygous missense mutations co-segregating with the syndrome. Subsequent analysis of 15 small ADLTE-affected families revealed four additional missense mutations. 3D modeling predicted that all mutations have structural effects on protein-domain folding. Overall, RELN mutations occurred in 7/40 (17.5%) ADLTE-affected families. RELN encodes a secreted protein, Reelin, which has important functions in both the developing and adult brain and is also found in the blood serum. We show that ADLTE-related mutations significantly decrease serum levels of Reelin, suggesting an inhibitory effect of mutations on protein secretion. We also show that Reelin and LGI1 co-localize in a subset of rat brain neurons, supporting an involvement of both proteins in a common molecular pathway underlying ADLTE. Homozygous RELN mutations are known to cause lissencephaly with cerebellar hypoplasia. Our findings extend the spectrum of neurological disorders associated with RELN mutations and establish a link between RELN and LGI1, which play key regulatory roles in both the developing and adult brain.


Assuntos
Moléculas de Adesão Celular Neuronais/genética , Epilepsia do Lobo Frontal/genética , Epilepsia do Lobo Frontal/patologia , Proteínas da Matriz Extracelular/genética , Modelos Moleculares , Mutação de Sentido Incorreto/genética , Proteínas do Tecido Nervoso/genética , Serina Endopeptidases/genética , Transtornos do Sono-Vigília/genética , Transtornos do Sono-Vigília/patologia , Animais , Sequência de Bases , Moléculas de Adesão Celular Neuronais/sangue , Moléculas de Adesão Celular Neuronais/química , Moléculas de Adesão Celular Neuronais/metabolismo , Mapeamento Cromossômico , Exoma , Proteínas da Matriz Extracelular/sangue , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Imunofluorescência , Componentes do Gene , Humanos , Immunoblotting , Peptídeos e Proteínas de Sinalização Intercelular , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/sangue , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Linhagem , Polimorfismo de Nucleotídeo Único/genética , Conformação Proteica , Dobramento de Proteína , Proteínas/metabolismo , Ratos , Proteína Reelina , Análise de Sequência de DNA , Serina Endopeptidases/sangue , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo
6.
Epilepsy Behav ; 68: 103-107, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28142128

RESUMO

OBJECTIVE: To describe the clinical phenotype of 7 families with Autosomal Dominant Lateral Temporal Lobe Epilepsy (ADLTE) related to Reelin (RELN) mutations comparing the data with those observed in 12 LGI1-mutated pedigrees belonging to our series. METHODS: Out of 40 Italian families with ADLTE, collected by epileptologists participating in a collaborative study of the Commission for Genetics of the Italian League against Epilepsy encompassing a 14-year period (2000-2014), 7 (17.5%) were found to harbor heterozygous RELN mutations. The whole series also included 12 (30%) LGI1 mutated families and 21 (52.5%) non-mutated pedigrees. The clinical, neurophysiological, and neuroradiological findings of RELN and LGI1 mutated families were analyzed. RESULTS: Out of 28 affected individuals belonging to 7 RELN mutated families, 24 had sufficient clinical data available for the study. In these patients, the epilepsy onset occurred at a mean age of 20years, with focal seizures characterized by auditory auras in about 71% of the cases, associated in one-third of patients with aphasia, visual disturbances or other less common symptoms (vertigo or déjà-vu). Tonic-clonic seizures were reported by almost all patients (88%), preceded by typical aura in 67% of cases. Seizures were precipitated by environmental noises in 8% of patients and were completely or almost completely controlled by antiepileptic treatment in the vast majority of cases (96%). The interictal EEG recordings showed epileptiform abnormalities or focal slow waves in 80% of patients, localized over the temporal regions, with marked left predominance and conventional 1,5T MRI scans were not contributory. By comparing these findings with those observed in families with LGI1 mutations, we did not observe significant differences except for a higher rate of left-sided EEG abnormalities in the RELN group. SIGNIFICANCE: Heterozygous RELN mutations cause a typical ADLTE syndrome, indistinguishable from that associated with LGI1 mutations.


Assuntos
Moléculas de Adesão Celular Neuronais/genética , Epilepsia do Lobo Frontal/genética , Proteínas da Matriz Extracelular/genética , Mutação , Proteínas do Tecido Nervoso/genética , Fenótipo , Serina Endopeptidases/genética , Transtornos do Sono-Vigília/genética , Adulto , Epilepsia do Lobo Frontal/diagnóstico , Feminino , Humanos , Itália , Masculino , Linhagem , Proteína Reelina , Transtornos do Sono-Vigília/diagnóstico , Adulto Jovem
7.
Epilepsia ; 56(10): e168-71, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26216793

RESUMO

Mutations in the DEPDC5 (DEP domain-containing protein 5) gene are a major cause of familial focal epilepsy with variable foci (FFEVF) and are predicted to account for 12-37% of families with inherited focal epilepsies. To assess the clinical impact of DEPDC5 mutations in familial temporal lobe epilepsy, we screened a collection of Italian families with either autosomal dominant lateral temporal epilepsy (ADLTE) or familial mesial temporal lobe epilepsy (FMTLE). The probands of 28 families classified as ADLTE and 17 families as FMTLE were screened for DEPDC5 mutations by whole exome or targeted massive parallel sequencing. Putative mutations were validated by Sanger sequencing. We identified a DEPDC5 nonsense mutation (c.918C>G; p.Tyr306*) in a family with two affected members, clinically classified as FMTLE. The proband had temporal lobe seizures with prominent psychic symptoms (déjà vu, derealization, and forced thoughts); her mother had temporal lobe seizures, mainly featuring visceral epigastric auras and anxiety. In total, we found a single DEPDC5 mutation in one of (2.2%) 45 families with genetic temporal lobe epilepsy, a proportion much lower than that reported in other inherited focal epilepsies.


Assuntos
Epilepsia do Lobo Temporal/genética , Saúde da Família , Mutação/genética , Proteínas Repressoras/genética , Adulto , Análise Mutacional de DNA , Feminino , Proteínas Ativadoras de GTPase , Humanos , Itália , Masculino
8.
Eur Heart J ; 34(3): 201-10, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23136403

RESUMO

AIMS: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a major cause of juvenile sudden death and is characterized by fibro-fatty replacement of the right ventricle. Mutations in several genes encoding desmosomal proteins have been identified in ARVC. We speculated that αT-catenin, encoded by CTNNA3, might also carry mutations in ARVC patients. Alpha-T-catenin binds plakophilins and this binding contributes to the formation of the area composita, which strengthens cell-cell adhesion in contractile cardiomyocytes. METHODS AND RESULTS: We used denaturing high-performance liquid chromatography and direct sequencing to screen CTNNA3 in 76 ARVC patients who did not carry any mutations in the desmosomal genes commonly mutated in ARVC. Mutations c.281T > A (p.V94D) and c.2293_2295delTTG (p.del765L) were identified in two probands. They are located in important domains of αT-catenin. Yeast two-hybrid and cell transfection studies showed that the interaction between the p.V94D mutant protein and ß-catenin was affected, whereas the p.del765L mutant protein showed a much stronger dimerization potential and formed aggresomes in HEK293T cells. CONCLUSION: These findings might point to a causal relationship between CTNNA3 mutations and ARVC. This first report on the involvement of an area composita gene in ARVC shows that the pathogenesis of this disease extends beyond desmosomes. Since the frequency of CTNNA3 mutations in ARVC patients is not rare, systematic screening for this gene should be considered to improve the clinical management of ARVC families.


Assuntos
Displasia Arritmogênica Ventricular Direita/genética , Morte Súbita Cardíaca/etiologia , Deleção de Genes , Mutação de Sentido Incorreto/genética , alfa Catenina/genética , Adulto , Arritmias Cardíacas/genética , Displasia Arritmogênica Ventricular Direita/metabolismo , Estudos de Casos e Controles , Eletrocardiografia , Feminino , Heterozigoto , Humanos , Masculino , Linhagem , alfa Catenina/metabolismo
9.
Epilepsia Open ; 9(3): 1083-1087, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38654463

RESUMO

Familial epilepsy with auditory features (FEAF), previously known as autosomal-dominant lateral temporal lobe epilepsy (ADLTE) is a genetically heterogeneous syndrome, clinically characterized by focal seizures with prominent auditory symptoms. It is inherited with autosomal-dominant pattern with reduced penetrance (about 70%). Sporadic epilepsy with auditory features cases are more frequent and clinically indistinguishable from familial cases. One causal gene, MICAL-1, encodes MICAL-1, an intracellular multi-domain enzyme that is an important regulator of filamentous actin (F-actin) structures. Pathogenic variants in MICAL-1 account for approximately 7% of FEAF families. Here, we describe a de novo MICAL-1 pathogenic variant, p.Arg915Cys, in a sporadic case, an affected 21-year-old Italian man with no family history of epilepsy. Genetic testing was performed in the patient and his parents, using a next-generation sequencing panel. In cell-based assay, this variant significantly increased MICAL-1 oxidoreductase activity, which likely resulted in dysregulation of F-actin organization. This finding provides further support for a gain-of-function effect underlying MICAL-1-mediated epilepsy pathogenesis, as previously seen with other pathogenic variants. Furthermore, the case study provides evidence that de novo MICAL-1 pathogenic variants can occur in sporadic cases with epilepsy with auditory feature (EAF). PLAIN LANGUAGE SUMMARY: In this study, we report a new MICAL-1 pathogenic variant in a patient without family history for epilepsy, not inherited from his parents. MICAL-1 is a protein with enzymatic activity that reorganizes the structure of the cell. We proved the pathological effect of this variant by testing its enzymatic activity and found an increase of this activity. This result suggests that non-familial cases should be tested to find novel pathogenic variants in this gene.


Assuntos
Epilepsia do Lobo Temporal , Humanos , Masculino , Adulto Jovem , Epilepsia do Lobo Temporal/genética , Actinas/genética
10.
Epilepsia ; 54(7): 1288-97, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23621105

RESUMO

PURPOSE: In relatively small series, autosomal dominant lateral temporal epilepsy (ADLTE) has been associated with leucine-rich, glioma-inactivated 1 (LGI1) mutations in about 50% of the families, this genetic heterogeneity being probably caused by differences in the clinical characteristics of the families. In this article we report the overall clinical and genetic spectrum of ADLTE in Italy with the aim to provide new insight into its nosology and genetic basis. METHODS: In a collaborative study of the Commission of Genetics of the Italian League Against Epilepsy (LICE) encompassing a 10-year period (2000-2010), we collected 33 ADLTE families, selected on the basis of the following criteria: presence of at least two members concordant for unprovoked partial seizures with prominent auditory and or aphasic symptoms, absence of any known structural brain pathology or etiology, and normal neurologic examination. The clinical, neurophysiologic, and neuroradiologic findings of all patients were analyzed and a genealogic tree was built for each pedigree. The probands' DNA was tested for LGI1 mutations by direct sequencing and, if negative, were genotyped with single-nucleotide polymorphism (SNP) array to search for disease-linked copy-number variation CNV. The disease penetrance in mutated and nonmutated families was assessed as a proportion of obligate carriers who were affected. KEY FINDINGS: The 33 families included a total of 127 affected individuals (61 male, 66 female, 22 deceased). The age at onset ranged between 2 and 60 years (mean 18.7 years). Ninety-one patients (72%) had clear-cut focal (elementary, complex, or secondarily generalized) seizures, characterized by prominent auditory auras in 68% of the cases. Other symptoms included complex visual hallucinations, vertigo, and déjà vu. Aphasic seizures, associated or not with auditory features, were observed in 20% of the cases, whereas tonic-clonic seizures occurred in 86% of the overall series. Sudden noises could precipitate the seizures in about 20% of cases. Seizures, which usually occurred at a low frequency, were promptly controlled or markedly improved by antiepileptic treatment in the majority of patients. The interictal electroencephalography (EEG) studies showed the epileptiform temporal abnormalities in 62% of cases, with a slight predominance over the left region. Magnetic resonance imaging (MRI) or computerized tomography (CT) scans were negative. LGI1 mutations (missense in nine and a microdeletion in one) were found in only 10 families (30%). The patients belonging to the mutated and not mutated groups did not differ except for penetrance estimate, which was 61.3% and 35% in the two groups, respectively (chi-square, p = 0.017). In addition, the disease risk of members of families with mutations in LGI1 was three times higher than that of members of LGI1-negative families (odds ratio [OR] 2.94, confidence interval [CI] 1.2-7.21). SIGNIFICANCE: A large number of ADLTE families has been collected over a 10-year period in Italy, showing a typical and homogeneous phenotype. LGI1 mutations have been found in only one third of families, clinically indistinguishable from nonmutated pedigrees. The estimate of penetrance and OR, however, demonstrates a significantly lower penetrance rate and relative disease risk in non-LGI1-mutated families compared with LGI1-mutated pedigrees, suggesting that a complex inheritance pattern may underlie a proportion of these families.


Assuntos
Epilepsia do Lobo Temporal/genética , Saúde da Família , Genes Dominantes/genética , Mutação/genética , Penetrância , Proteínas/genética , Estimulação Acústica , Adolescente , Adulto , Idade de Início , Criança , Pré-Escolar , Análise Mutacional de DNA , Eletroencefalografia , Epilepsia do Lobo Temporal/fisiopatologia , Feminino , Genótipo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Itália , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Fenótipo , Estudos Retrospectivos , Adulto Jovem
11.
Curr Neurol Neurosci Rep ; 12(4): 445-55, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22618127

RESUMO

Genetic factors are likely to play a major role in many epileptic conditions, spanning from classical idiopathic (genetic) generalized epilepsies to epileptic encephalopathies and focal epilepsies. In this review we describe the genetic advances in progressive myoclonus epilepsies, which are strictly monogenic disorders, genetic generalized epilepsies, mostly exhibiting complex genetic inheritance, and SCN1A-related phenotypes, namely genetic generalized epilepsy with febrile seizure plus and Dravet syndrome. Particular attention is devoted to a form of familial focal epilepsies, autosomal-dominant lateral temporal epilepsy, which is a model of non-ion genetic epilepsies. This condition is associated with mutations of the LGI1 gene, whose protein is secreted from the neurons and exerts its action on a number of targets, influencing cortical development and neuronal maturation.


Assuntos
Epilepsia/genética , Mutação/genética , Animais , Genes Dominantes/genética , Humanos , Fenótipo
12.
Exp Cell Res ; 315(6): 915-27, 2009 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-19371636

RESUMO

Skeletal muscle regeneration relies on satellite cells, a population of myogenic precursors. Inflammation also plays a determinant role in the process, as upon injury, macrophages are attracted by the damaged myofibers and the activated satellite cells and act as key elements of dynamic muscle supportive stroma. Yet, it is not known how macrophages interact with the more profound stem cells of the satellite cell niche. Here we show that in the presence of a murine macrophage conditioned medium (mMCM) a subpopulation of multipotent cells could be selected and expanded from adult rat muscle. These cells were small, round, poorly adhesive, slow-growing and showed mesenchymal differentiation plasticity. At the same time, mMCM showed clear myogenic capabilities, as experiments with satellite cells mechanically isolated from suspensions of single myofibers showed that the macrophagic factors inhibited their tendency to shift towards adipogenesis. In vivo, intramuscular administrations of concentrated mMCM in a rat model of extensive surgical ablation dramatically improved muscle regeneration. Altogether, these findings suggest that macrophagic factors could be of great help in developing therapeutic protocols with myogenic stem cells.


Assuntos
Meios de Cultivo Condicionados/química , Macrófagos/metabolismo , Células-Tronco Multipotentes/fisiologia , Desenvolvimento Muscular/fisiologia , Músculo Esquelético , Células Satélites de Músculo Esquelético/fisiologia , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Proliferação de Células , Humanos , Macrófagos/citologia , Masculino , Camundongos , Células-Tronco Multipotentes/citologia , Músculo Esquelético/citologia , Músculo Esquelético/fisiologia , Ratos , Ratos Wistar , Regeneração/fisiologia , Células Satélites de Músculo Esquelético/citologia
13.
Epileptic Disord ; 22(4): 443-448, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32723706

RESUMO

Reelin mutations are responsible for a minority of families with autosomal dominant lateral temporal lobe epilepsy. Here, we report a novel nuclear family with distinct clinical and neuroradiological findings. We studied the proband and her mother by means of EEG, video-EEG, 3T MRI, FDG-PET and genetic testing. Both patients had a focal drug-resistant epilepsy with onset at the age of 16 and focal seizures with typical auditory features combined with fear, followed by loss of contact or evolving to bilateral tonic-clonic seizures. The proband's ictal EEG showed clear left temporal seizure onset, and cerebral MRI revealed subtle left temporal changes (mild hypotrophy, slight blurring of the white and grey matter and hyperintensity) with corresponding left temporal mesial focal hypometabolism on FDG-PET. Genetic testing identified a missense variant, c.6631C>T (p.Arg2211Cys), in reelin repeat #5 in both patients, which markedly affected the secretion of the protein. The data from this family support previous findings indicating that reelin mutations are a cause of autosomal dominant lateral temporal lobe epilepsy which has a clinical spectrum that may also encompass drug-resistant epilepsy associated with mild MRI temporal changes.


Assuntos
Moléculas de Adesão Celular Neuronais/genética , Epilepsia do Lobo Frontal/diagnóstico , Epilepsia do Lobo Frontal/genética , Proteínas da Matriz Extracelular/genética , Proteínas do Tecido Nervoso/genética , Serina Endopeptidases/genética , Transtornos do Sono-Vigília/diagnóstico , Transtornos do Sono-Vigília/genética , Adulto , Idoso , Eletroencefalografia , Epilepsia do Lobo Frontal/patologia , Epilepsia do Lobo Frontal/fisiopatologia , Feminino , Humanos , Imageamento por Ressonância Magnética , Linhagem , Proteína Reelina , Transtornos do Sono-Vigília/patologia , Transtornos do Sono-Vigília/fisiopatologia
14.
Cardiovasc Res ; 115(4): 739-751, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30304392

RESUMO

AIMS: Arrhythmogenic cardiomyopathy (AC) is one of the most common inherited cardiomyopathies, characterized by progressive fibro-fatty replacement in the myocardium. Clinically, AC manifests itself with ventricular arrhythmias, syncope, and sudden death and shows wide inter- and intra-familial variability. Among the causative genes identified so far, those encoding for the desmosomal proteins plakophilin-2 (PKP2), desmoplakin (DSP), and desmoglein-2 (DSG2) are the most commonly mutated. So far, little is known about the molecular mechanism(s) behind such a varied spectrum of phenotypes, although it has been shown that the causative mutations not only lead to structural abnormalities but also affect the miRNA profiling of cardiac tissue. Here, we aimed at studying the pathogenic effects of a nonsense mutation of the desmoglein-2 gene, both at the structural level and in terms of miRNA expression pattern. METHODS AND RESULTS: We generated transgenic mice with cardiomyocyte-specific overexpression of a FLAG-tagged human desmoglein-2 harbouring the Q558* nonsense mutation found in an AC patient. The hearts of these mice showed signs of fibrosis, decrease in desmosomal size and number, and reduction of the Wnt/ß-catenin signalling. Genome-wide RNA-Seq performed in Tg-hQ hearts and non-transgenic hearts revealed that 24 miRNAs were dysregulated in transgenic animals. Further bioinformatic analyses for selected miRNAs suggested that miR-217-5p, miR-499-5p, and miR-708-5p might be involved in the pathogenesis of the disease. CONCLUSION: Down-regulation of the canonical Wnt/ß-catenin signalling might be considered a common key event in the AC pathogenesis. We identified the miRNA signature in AC hearts, with miR-708-5p and miR-217-5p being the most up-regulated and miR-499-5p the most down-regulated miRNAs. All of them were predicted to be involved in the regulation of the Wnt/ß-catenin pathway and might reveal the potential pathophysiology mechanisms of AC, as well as be useful as therapeutic targets for the disease.


Assuntos
Displasia Arritmogênica Ventricular Direita/genética , Códon sem Sentido , Desmogleína 2/genética , MicroRNAs/genética , Miocárdio/metabolismo , Via de Sinalização Wnt/genética , Animais , Displasia Arritmogênica Ventricular Direita/metabolismo , Displasia Arritmogênica Ventricular Direita/patologia , Modelos Animais de Doenças , Fibrose , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Predisposição Genética para Doença , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/metabolismo , Miocárdio/ultraestrutura , Fenótipo , Transcriptoma
15.
Clin Neurol Neurosurg ; 170: 27-33, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29723732

RESUMO

OBJECTIVES: The Leucine-rich glioma inactivated 1 (LGI1) protein is thought to be implicated in malignant progression of glioma tumors, and mutations in the encoding gene, LGI1, cause autosomal dominant lateral temporal epilepsy, a genetic focal epilepsy syndrome. The aim of this study was to investigate the possible involvement of LGI1 in high-grade glioma-associated epilepsy by analyzing its expression in tumor specimens of patients with and without epilepsy and by searching for LGI1 autoantibodies in the sera these patients. PATIENTS AND METHODS: We examined tumor tissue samples from 24 patients with high-grade gliomas (12 with and 12 without epilepsy) by immunoblot and detected variable amounts of LGI1 in tumor tissues from 9/24 (37%) patients. RESULTS: LGI1 was detected in 7/12 (58%) patients with epilepsy and in 2/12 (16%) patients without epilepsy (p = 0.0894; Fisher's exact test). Moreover, testing blood sera of five patients for antibodies against LGI1 revealed LGI1 autoantibodies in two patients, both suffering from epilepsy and expressing LGI1 in tumor tissue. CONCLUSION: Our findings suggest that there may be a preferential expression of LGI1 in high-grade glioma tumors of patients with epilepsy. We also unveil the presence of serum LGI1 autoantibodies in some patients with high-grade gliomas, where they might play an epileptogenic role.


Assuntos
Astrocitoma/sangue , Autoanticorpos/sangue , Neoplasias Encefálicas/sangue , Epilepsia do Lobo Temporal/sangue , Glioblastoma/sangue , Proteínas/metabolismo , Adulto , Idoso , Astrocitoma/diagnóstico , Astrocitoma/genética , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/genética , Epilepsia do Lobo Temporal/diagnóstico , Epilepsia do Lobo Temporal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Glioblastoma/diagnóstico , Glioblastoma/genética , Glioma/sangue , Glioma/diagnóstico , Glioma/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Pessoa de Meia-Idade , Proteínas/genética , Adulto Jovem
16.
Epilepsy Res ; 139: 51-53, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29179159

RESUMO

Autosomal dominant epilepsy with auditory features (ADEAF) is clinically characterized by focal seizures with prominent auditory or aphasic auras and absence of structural brain abnormalities. Mutations in LGI1 and RELN genes account for the disorder in about 50% of ADEAF families. In a recent paper, a heterozygous intragenic deletion in the CNTNAP2 gene has been associated to ADEAF in a single family. We screened 28 ADEAF families for mutations in CNTNAP2 by next generation sequencing and copy number variation analyses and found no likely pathogenic mutations segregating with the disease. CNTNAP2 should be screened in genetically unsolved ADEAF families, but causative mutations are expected to be infrequent in this gene.


Assuntos
Epilepsia do Lobo Temporal/genética , Proteínas de Membrana/genética , Mutação , Proteínas do Tecido Nervoso/genética , Variações do Número de Cópias de DNA , Família , Predisposição Genética para Doença , Humanos , Proteína Reelina
17.
Arthritis Res Ther ; 17: 93, 2015 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-25888769

RESUMO

INTRODUCTION: Mutations in the TNFRSF1A gene, encoding tumor necrosis factor receptor 1 (TNF-R1), are associated with the autosomal dominant autoinflammatory disorder, called TNF receptor associated periodic syndrome (TRAPS). TRAPS is clinically characterized by recurrent episodes of long-lasting fever and systemic inflammation. A novel mutation (c.262 T > C; S59P) in the TNFRSF1A gene at residue 88 of the mature protein was recently identified in our laboratory in an adult TRAPS patient. The aim of this study was to functionally characterize this novel TNFRSF1A mutation evaluating its effects on the TNF-R1-associated signaling pathways, firstly NF-κB, under particular conditions and comparing the results with suitable control mutations. METHODS: HEK-293 cell line was transfected with pCMV6-AC construct expressing wild-type (WT) or c.262 T > C (S59P), c.362G > A (R92Q), c.236C > T (T50M) TNFRSF1A mutants. Peripheral blood mononuclear cells (PBMCs) were instead isolated from two TRAPS patients carrying S59P and R92Q mutations and from five healthy subjects. Both transfected HEK-293 and PBMCs were stimulated with tumor necrosis factor (TNF) or interleukin 1ß (IL-1ß) to evaluate the expression of TNF-R1, the activation of TNF-R1-associated downstream pathways and the pro-inflammatory cytokines by means of immunofluorescent assay, array-based technique, immunoblotting and immunometric assay, respectively. RESULTS: TNF induced cytoplasmic accumulation of TNF-R1 in all mutant cells. Furthermore, all mutants presented a particular set of active TNF-R1 downstream pathways. S59P constitutively activated IL-1ß, MAPK and SRC/JAK/STAT3 pathways and inhibited apoptosis. Also, NF-κB pathway involvement was demonstrated in vitro by the enhancement of p-IκB-α and p65 nuclear subunit of NF-κB expression in all mutants in the presence of TNF or IL-1ß stimulation. These in vitro results correlated with patients' data from PBMCs. Concerning the pro-inflammatory cytokines secretion, mainly IL-1ß induced a significant and persistent enhancement of IL-6 and IL-8 in PBMCs carrying the S59P mutation. CONCLUSIONS: The novel S59P mutation leads to defective cellular trafficking and to constitutive activation of TNF-R1. This mutation also determines constitutive activation of the IL-1R pathway, inhibition of apoptosis and enhanced and persistent NF-κB activation and cytokine secretion in response to IL-1ß stimulation.


Assuntos
Febre Familiar do Mediterrâneo/genética , Predisposição Genética para Doença , Mutação , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/genética , Adulto , Fatores Etários , Idoso , Apoptose/genética , Estudos de Casos e Controles , Células Cultivadas , Febre Familiar do Mediterrâneo/fisiopatologia , Células HEK293 , Humanos , Immunoblotting/métodos , Itália , Masculino , NF-kappa B/metabolismo , Reação em Cadeia da Polimerase/métodos , Valores de Referência , Medição de Risco , Estudos de Amostragem , Transdução de Sinais
18.
Epilepsy Res ; 110: 132-8, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25616465

RESUMO

PURPOSE: Autosomal dominant lateral temporal epilepsy (ADLTE) is a genetic focal epilepsy syndrome characterized by prominent auditory or aphasic symptoms. Mutations in LGI1 account for less than 50% of ADLTE families. We assessed the impact of LGI1 microrearrangements in a collection of ADLTE families and sporadic lateral temporal epilepsy (LTE) patients, and investigated novel ADLTE and LTE patients. METHODS: Twenty-four ADLTE families and 140 sporadic LTE patients with no evidence of point mutations in LGI1 were screened for copy number alterations using multiplex ligation-dependent probe amplification (MLPA). Newly ascertained familial and sporadic LTE patients were clinically investigated, and interictal EEG and MRI findings were obtained; probands were tested for LGI1 mutations by direct exon sequencing or denaturing high performance liquid chromatography. RESULTS: We identified a novel microdeletion spanning LGI1 exon 2 in a family with two affected members, both presenting focal seizures with visual symptoms. Also, we identified a novel LGI1 missense mutation (c.1118T > C; p.L373S) in a newly ascertained family with focal seizures with prominent visual auras, and another missense mutation (c.856T > C; p.C286R) in a sporadic patient with auditory seizures. CONCLUSIONS: We describe two novel ADLTE families with predominant visual auras segregating pathogenic LGI1 mutations. These findings support the notion that, in addition to auditory symptoms, other types of auras can be found in patients carrying LGI1 mutations. The identification of a novel microdeletion in LGI1, the second so far identified, suggests that LGI1 microrearrangements may not be exceptional.


Assuntos
Epilepsia do Lobo Frontal/genética , Epilepsia do Lobo Frontal/fisiopatologia , Epilepsia do Lobo Temporal/genética , Proteínas/genética , Transtornos do Sono-Vigília/genética , Transtornos do Sono-Vigília/fisiopatologia , Idoso , Encéfalo/patologia , Encéfalo/fisiopatologia , Variações do Número de Cópias de DNA , Epilepsia do Lobo Frontal/patologia , Epilepsia do Lobo Temporal/patologia , Epilepsia do Lobo Temporal/fisiopatologia , Família , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Linhagem , Deleção de Sequência , Transtornos do Sono-Vigília/patologia , Adulto Jovem
19.
Epilepsy Res ; 108(2): 232-40, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24315020

RESUMO

PURPOSE: To describe the clinical findings in a family with a benign form of mesial temporal lobe epilepsy and to identify the causative genetic factors. METHODS: All participants were personally interviewed and underwent neurologic examination. The affected subjects underwent EEG and most of them neuroradiological examinations (MRI). All family members were genotyped with the HumanCytoSNP-12 v1.0 beadchip and linkage analysis was performed with Merlin and Simwalk2 programs. Exome sequencing was performed on HiSeq2000, after exome capture with SureSelect 50 Mb kit v2.0. RESULTS: The family had 6 members with temporal lobe epilepsy. Age at seizure onset ranged from 8 to 13 years. Five patients had epigastric auras often associated to oro-alimentary automatic activity, 3 patients presented loss of contact, and 2 experienced secondary generalizations. Febrile seizures occurred in 2 family members, 1 of whom also had temporal lobe epilepsy. EEG showed focal slow waves and epileptic abnormalities on temporal regions in 1 patient and was normal in the other affected individuals. MRI was normal in all temporal lobe epilepsy patients. We performed single nucleotide polymorphism-array linkage analysis of the family and found suggestive evidence of linkage (LOD score=2.106) to a region on chromosome 3q26. Haplotype reconstruction supported the linkage data and showed that the majority of unaffected family members carried the haplotype at risk. Whole exome sequencing failed to identify pathogenic mutations in genes of the candidate region. CONCLUSIONS: Our data suggest the existence of a novel locus for benign familial mesial temporal lobe epilepsy on chromosome 3q26. Our failure to identify pathogenic mutations in genes of this region may be due to limitations of the exome sequencing technology.


Assuntos
Cromossomos Humanos Par 3/genética , Epilepsia do Lobo Temporal/congênito , Ligação Genética/genética , Adolescente , Criança , Epilepsia do Lobo Temporal/diagnóstico , Epilepsia do Lobo Temporal/genética , Feminino , Humanos , Masculino , Linhagem
20.
Am J Cardiol ; 111(3): 400-5, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23168288

RESUMO

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart muscle disease characterized by fibrofatty replacement of the myocardium and ventricular arrhythmias, associated with mutations in the desmosomal genes. Only a missense mutation in the DES gene coding for desmin, the intermediate filament protein expressed by cardiac and skeletal muscle cells, has been recently associated with ARVC. We screened 91 ARVC index cases (53 negative for mutations in desmosomal genes and an additional 38 carrying desmosomal gene mutations) for DES mutations. Two rare missense variants were identified. The heterozygous p.K241E substitution was detected in 1 patient affected with a severe form of ARVC who also carried the p.T816RfsX10 mutation in plakophilin-2 gene. This DES substitution, showing an allele frequency of <0.01 in the control population, is predicted to cause an intolerant amino acid change in a highly conserved protein domain. Thus, it can be considered a rare variant with a possible modifier effect on the phenotypic expression of the concomitant mutation. The previously known p.A213V substitution was identified in 1 patient with ARVC who was negative for mutations in the desmosomal genes. Because a greater prevalence of p.A213V has been reported in patients with heart dilation than in control subjects, the hypothesis that this rare variant could have an unfavorable effect on cardiac remodeling cannot be ruled out. In conclusion, our data help to establish that, in the absence of skeletal muscle involvement suggestive of a desminopathy, the probability of DES mutations in ARVC is very low. These findings have important implications in the mutation screening strategy for patients with ARVC.


Assuntos
Displasia Arritmogênica Ventricular Direita/genética , DNA/genética , Desmina/genética , Mutação , Displasia Arritmogênica Ventricular Direita/metabolismo , Displasia Arritmogênica Ventricular Direita/fisiopatologia , Cromatografia Líquida de Alta Pressão , Análise Mutacional de DNA , Desmina/metabolismo , Eletrocardiografia , Feminino , Humanos , Masculino , Miocárdio/metabolismo , Linhagem , Fenótipo
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