RESUMO
PURPOSE: EFHC1 variants are the most common mutations in inherited myoclonic and grand mal clonic-tonic-clonic (CTC) convulsions of juvenile myoclonic epilepsy (JME). We reanalyzed 54 EFHC1 variants associated with epilepsy from 17 cohorts based on National Human Genome Research Institute (NHGRI) and American College of Medical Genetics and Genomics (ACMG) guidelines for interpretation of sequence variants. METHODS: We calculated Bayesian LOD scores for variants in coinheritance, unconditional exact tests and odds ratios (OR) in case-control associations, allele frequencies in genome databases, and predictions for conservation/pathogenicity. We reviewed whether variants damage EFHC1 functions, whether efhc1-/- KO mice recapitulate CTC convulsions and "microdysgenesis" neuropathology, and whether supernumerary synaptic and dendritic phenotypes can be rescued in the fly model when EFHC1 is overexpressed. We rated strengths of evidence and applied ACMG combinatorial criteria for classifying variants. RESULTS: Nine variants were classified as "pathogenic," 14 as "likely pathogenic," 9 as "benign," and 2 as "likely benign." Twenty variants of unknown significance had an insufficient number of ancestry-matched controls, but ORs exceeded 5 when compared with racial/ethnic-matched Exome Aggregation Consortium (ExAC) controls. CONCLUSIONS: NHGRI gene-level evidence and variant-level evidence establish EFHC1 as the first non-ion channel microtubule-associated protein whose mutations disturb R-type VDCC and TRPM2 calcium currents in overgrown synapses and dendrites within abnormally migrated dislocated neurons, thus explaining CTC convulsions and "microdysgenesis" neuropathology of JME.Genet Med 19 2, 144-156.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Epilepsia Mioclônica Juvenil/genética , Convulsões/genética , Animais , Dendritos/patologia , Exoma , Frequência do Gene , Humanos , Camundongos , Camundongos Knockout , Mutação , Epilepsia Mioclônica Juvenil/fisiopatologia , National Human Genome Research Institute (U.S.) , Neurônios/patologia , Linhagem , Polimorfismo de Nucleotídeo Único , Convulsões/fisiopatologia , Sinapses/patologia , Estados UnidosRESUMO
Lafora disease (LD) is an autosomal recessive, always fatal progressive myoclonus epilepsy with rapid cognitive and neurologic deterioration. One of the pathological hallmarks of LD is the presence of cytoplasmic PAS+polyglucosan inclusions called Lafora bodies (LBs). Current clinical and neuropathological views consider LBs to be the cause of neurological derangement of patients. A systematic study of the ontogeny and structural features of the LBs has not been done in the past. Therefore, we undertook a detailed microscopic analysis of the neuropile of a Laforin-deficient (epm2a-/-) mouse model. Wild type and epm2a-/- mice were sacrificed at different ages and their encephalon processed for light microscopy. Luxol-fast-blue, PAS, Bielschowski techniques, as well as immunocytochemistry (TUNEL, Caspase-3, Apaf-1, Cytochrome-C and Neurofilament L antibodies) were used. Young null mice (11 days old) showed necrotic neuronal death in the absence of LBs. Both cell death and LBs showed a progressive increment in size and number with age. Type I LBs emerged at two weeks of age and were distributed in somata and neurites. Type II LBs appeared around the second month of age and always showed a complex architecture and restricted to neuronal somata. Their number was considerably less than type I LBs. Bielschowski method showed neurofibrillary degeneration and senile-like plaques. These changes were more prominent in the hippocampus and ventral pons. Neurofibrillary tangles were already present in 11 days-old experimental animals, whereas senile-like plaques appeared around the third to fourth month of life. The encephalon of null mice was not uniformly affected: Diencephalic structures were spared, whereas cerebral cortex, basal ganglia, pons, hippocampus and cerebellum were notoriously affected. This uneven distribution was present even within the same structure, i.e., hippocampal sectors. Of special relevance, was the observation of the presence of immunoreactivity to neurofilament L on the external rim of type II LBs. Perhaps, type II LB is not the end point of a metabolic abnormality. Instead, we suggest that type II LB is a highly specialized structural and functional entity that emerges as a neuronal response to major carbohydrate metabolism impairment. Early necrotic cell death, neurocytoskeleton derangement, different structural and probably functional profiles for both forms of LBs, a potential relationship between the external rim of the LB type II and the cytoskeleton and an uneven distribution of these abnormalities indicate that LD is both a complex neurodegenerative disease and a glycogen metabolism disorder. Our findings are critical for future studies on disease mechanisms and therapies for LD. Interestingly, the neurodegenerative changes observed in this LD model can also be useful for understanding the process of dementia.
Assuntos
Citoesqueleto/patologia , Fosfatases de Especificidade Dupla/genética , Doença de Lafora/genética , Doença de Lafora/patologia , Degeneração Neural/patologia , Neurônios/patologia , Animais , Gânglios da Base/patologia , Tronco Encefálico/patologia , Citoplasma/patologia , Modelos Animais de Doenças , Feminino , Hipocampo/patologia , Doença de Lafora/metabolismo , Masculino , Camundongos , Camundongos Mutantes , Degeneração Neural/genética , Degeneração Neural/metabolismo , Emaranhados Neurofibrilares/patologia , Neurópilo/patologia , Fenótipo , Placa Amiloide/patologia , Proteínas Tirosina Fosfatases não Receptoras , Área Tegmentar Ventral/patologiaRESUMO
PURPOSE: Juvenile myoclonic epilepsy (JME) accounts for 3 to 12% of all epilepsies. In 2004, we identified a mutation-harboring Mendelian gene that encodes a protein with one EF-hand motif (EFHC1) in chromosome 6p12. We observed one doubly heterozygous and three heterozygous missense mutations in EFHC1 segregating as an autosomal dominant gene with 21 affected members of six Hispanic JME families from California and Mexico. In 2006, similar and three novel missense mutations were reported in sporadic and familial Caucasian JME from Italy and Austria. In this study, we asked if coding single nucleotide polymorphisms (SNPs) of EFHC1 also contribute as susceptibility alleles to JME with complex genetics. METHODS: We screened using denaturing high-performance liquid chromatography (DHPLC) and then directly sequenced the 11 exons of EFHC1 in 130 unrelated JME probands, their 352 family members, and seven exons of EFHC1 in 400-614 ethnically matched controls. We carried out case-control association studies between 124 unrelated Hispanic JME probands and 552-614 ethnically matched controls using four SNPs, rs3804506, rs3804505, rs1266787, and rs17851770. We also performed family-based association on SNPs rs3804506 and rs3804505 in 84 complete JME families using the Family-Based Association Test (FBAT) program. RESULTS: We found no statistically significant differences between JME probands and controls in case-control association and no genetic transmission disequilibria in family-based association for the tested SNPs. In addition, we identified four new DNA variants in the coding region of EFHC1. CONCLUSION: The four coding SNPs, rs3804506, rs3804505, rs1266787, and rs17851770, of EFHC1 may not be susceptibility alleles for JME.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Epilepsia Mioclônica Juvenil/genética , Polimorfismo de Nucleotídeo Único/genética , Estudos de Casos e Controles , Análise Mutacional de DNA/métodos , Éxons/genética , Saúde da Família , Feminino , Humanos , Masculino , Dados de Sequência MolecularRESUMO
INTRODUCTION: After unilateral dopamine depletion, some ipsilateral alterations occur and the contralateral structure has been utilized as control. OBJECTIVE: Our aim is to analyse the evolution of the ultrastructural alterations of the ipsilateral and contralateral striata of the 6-hydroxydopamine lesioned rats to demonstrate that the contralateral striatum should not be used as control structure. METHODS: After the surgery and the rotation behavior evaluation, animals were killed from 3 to 120 days after lesioning, and their striata were compared with those of aged rats. RESULTS: The ultrastructural analysis shows increased diameter of the synaptic ending in ipsilateral (since the third day) and contralateral striata (since day 30) and an increase in perforated synaptic contacts. CONCLUSION: Our data suggest that the contralateral striatum should not be taken as control structure at least after 20-30 days after lesioning, as the alterations found here may result in wrong interpretations when comparing with the ipsilateral-lesioned one.
Assuntos
Corpo Estriado/metabolismo , Dopamina/metabolismo , Lateralidade Funcional/fisiologia , Neurópilo/patologia , Síndromes Neurotóxicas/metabolismo , Síndromes Neurotóxicas/patologia , Síndromes Neurotóxicas/fisiopatologia , Adrenérgicos , Animais , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/ultraestrutura , Modelos Animais de Doenças , Lateralidade Funcional/efeitos dos fármacos , Masculino , Microscopia Eletrônica de Transmissão/métodos , Neurópilo/ultraestrutura , Síndromes Neurotóxicas/etiologia , Oxidopamina/toxicidade , Ratos , Ratos Wistar , Sinapses/efeitos dos fármacos , Sinapses/ultraestrutura , Fatores de TempoRESUMO
Childhood absence epilepsy (CAE) accounts for 10% to 12% of epilepsy in children under 16 years of age. We screened for mutations in the GABA(A) receptor (GABAR) beta 3 subunit gene (GABRB3) in 48 probands and families with remitting CAE. We found that four out of 48 families (8%) had mutations in GABRB3. One heterozygous missense mutation (P11S) in exon 1a segregated with four CAE-affected persons in one multiplex, two-generation Mexican family. P11S was also found in a singleton from Mexico. Another heterozygous missense mutation (S15F) was present in a singleton from Honduras. An exon 2 heterozygous missense mutation (G32R) was present in two CAE-affected persons and two persons affected with EEG-recorded spike and/or sharp wave in a two-generation Honduran family. All mutations were absent in 630 controls. We studied functions and possible pathogenicity by expressing mutations in HeLa cells with the use of Western blots and an in vitro translation and translocation system. Expression levels did not differ from those of controls, but all mutations showed hyperglycosylation in the in vitro translation and translocation system with canine microsomes. Functional analysis of human GABA(A) receptors (alpha 1 beta 3-v2 gamma 2S, alpha 1 beta 3-v2[P11S]gamma 2S, alpha 1 beta 3-v2[S15F]gamma 2S, and alpha 1 beta 3-v2[G32R]gamma 2S) transiently expressed in HEK293T cells with the use of rapid agonist application showed that each amino acid transversion in the beta 3-v2 subunit (P11S, S15F, and G32R) reduced GABA-evoked current density from whole cells. Mutated beta 3 subunit protein could thus cause absence seizures through a gain in glycosylation of mutated exon 1a and exon 2, affecting maturation and trafficking of GABAR from endoplasmic reticulum to cell surface and resulting in reduced GABA-evoked currents.
Assuntos
Epilepsia Tipo Ausência/genética , Epilepsia Tipo Ausência/fisiopatologia , Mutação de Sentido Incorreto , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Ácido gama-Aminobutírico/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Criança , Pré-Escolar , Análise Mutacional de DNA , DNA Complementar/genética , Eletroencefalografia , Feminino , Ligação Genética , Genótipo , Glicosilação , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Fenótipo , Receptores de GABA-A/química , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
The 2001 classification subcommittee of the International League Against Epilepsy (ILAE) proposed to 'group JME, juvenile absence epilepsy, and epilepsy with tonic clonic seizures only under the sole heading of idiopathic generalized epilepsies (IGE) with variable phenotype'. The implication is that juvenile myoclonic epilepsy (JME) does not exist as the sole phenotype of family members and that it should no longer be classified by itself or considered a distinct disease entity. Although recognized as a common form of epilepsy and presumed to be a lifelong trait, a long-term follow-up of JME has not been performed. To address these two issues, we studied 257 prospectively ascertained JME patients and encountered four groups: (i) classic JME (72%), (ii) CAE (childhood absence epilepsy) evolving to JME (18%), (iii) JME with adolescent absence (7%), and (iv) JME with astatic seizures (3%). We examined clinical and EEG phenotypes of family members and assessed clinical course over a mean of 11 +/- 6 years and as long as 52 years. Forty per cent of JME families had JME as their sole clinical phenotype. Amongst relatives of classic JME families, JME was most common (40%) followed by grand mal (GM) only (35%). In contrast, 66% of families with CAE evolving to JME expressed the various phenotypes of IGE in family members. Absence seizures were more common in family members of CAE evolving to JME than in those of classic JME families (P < 0.001). Female preponderance, maternal transmission and poor response to treatment further characterized CAE evolving to JME. Only 7% of those with CAE evolving to JME were seizure-free compared with 58% of those with classic JME (P < 0.001), 56% with JME plus adolescent pyknoleptic absence and 62% with JME plus astatic seizures. Long-term follow-up (1-40 years for classic JME; 5-52 years for CAE evolving to JME, 5-26 years for JME with adolescent absence and 3-18 years for JME with astatic seizures) indicates that all subsyndromes are chronic and perhaps lifelong. Seven chromosome loci, three epilepsy-causing mutations and two genes with single nucleotide polymorphisms (SNPs) associating with JME reported in literature provide further evidence for JME as a distinct group of diseases.
Assuntos
Epilepsia Mioclônica Juvenil/genética , Adolescente , Adulto , Idade de Início , Criança , Doença Crônica , Progressão da Doença , Eletroencefalografia , Família , Feminino , Seguimentos , Humanos , Masculino , Epilepsia Mioclônica Juvenil/classificação , Fenótipo , Polimorfismo de Nucleotídeo Único , Prognóstico , Fatores SexuaisRESUMO
Idiopathic generalized epilepsies (IGEs) comprise at least 40% of epilepsies in the United States, 20% in Mexico, and 8% in Central America. Here, we review seizure phenotypes across IGE syndromes, their response to treatment and advances in molecular genetics that influence nosology. Our review included the Medline database from 1945 to 2005 and our prospectively collected Genetic Epilepsy Studies (GENESS) Consortium database. Generalized seizures occur with different and similar semiologies, frequencies, and patterns, ages at onset, and outcomes in different IGEs, suggesting common neuroanatomical pathways for seizure phenotypes. However, the same seizure phenotypes respond differently to the same treatments in different IGEs, suggesting different molecular defects across syndromes. De novo mutations in SCN1A in sporadic Dravet syndrome and germline mutations in SCN1A, SCN1B, and SCN2A in generalized epilepsies with febrile seizures plus have unraveled the heterogenous myoclonic epilepsies of infancy and early childhood. Mutations in GABRA1, GABRG2, and GABRB3 are associated with absence seizures, while mutations in CLCN2 and myoclonin/EFHC1 substantiate juvenile myoclonic epilepsy as a clinical entity. Refined understanding of seizure phenotypes, their semiology, frequencies, and patterns together with the identification of molecular lesions in IGEs continue to accelerate the development of molecular epileptology.
Assuntos
Epilepsia Generalizada/classificação , Epilepsia Generalizada/genética , Adolescente , Adulto , Distribuição por Idade , Anticonvulsivantes/uso terapêutico , Criança , Pré-Escolar , Epilepsia Generalizada/epidemiologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Biologia Molecular , Mutação/genética , Fenótipo , Prognóstico , SíndromeAssuntos
Epilepsia Tipo Ausência/complicações , Saúde da Família , Epilepsia Mioclônica Juvenil/etiologia , Adolescente , Adulto , Idade de Início , Criança , Pré-Escolar , Diagnóstico por Imagem , Eletroencefalografia/métodos , Epilepsia Tipo Ausência/genética , Feminino , Humanos , Lactente , Escore Lod , Masculino , Epilepsia Mioclônica Juvenil/genética , Exame Neurológico , RiscoAssuntos
Saúde da Família , Epilepsia Mioclônica Juvenil/genética , Adolescente , Adulto , Idade de Início , Mapeamento Cromossômico/métodos , Demografia , Eletroencefalografia/métodos , Epilepsia/complicações , Epilepsia Tipo Ausência/complicações , Epilepsia Tipo Ausência/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Epilepsia Mioclônica Juvenil/classificação , Epilepsia Mioclônica Juvenil/etiologia , Fenótipo , Risco , Fatores SexuaisRESUMO
Mutations in the EPM2A gene encoding a dual-specificity phosphatase (laforin) cause Lafora disease (LD), a progressive and invariably fatal epilepsy with periodic acid-Schiff-positive (PAS+) cytoplasmic inclusions (Lafora bodies) in the central nervous system. To study the pathology of LD and the functions of laforin, we disrupted the Epm2a gene in mice. At two months of age, homozygous null mutants developed widespread degeneration of neurons, most of which occurred in the absence of Lafora bodies. Dying neurons characteristically exhibit swelling in the endoplasmic reticulum, Golgi networks and mitochondria in the absence of apoptotic bodies or fragmentation of DNA. As Lafora bodies become more prominent at 4-12 months, organelles and nuclei are disrupted. The Lafora bodies, present both in neuronal and non-neural tissues, are positive for ubiquitin and advanced glycation end-products only in neurons, suggesting different pathological consequence for Lafora inclusions in neuronal tissues. Neuronal degeneration and Lafora inclusion bodies predate the onset of impaired behavioral responses, ataxia, spontaneous myoclonic seizures and EEG epileptiform activity. Our results suggest that LD is a primary neurodegenerative disorder that may utilize a non-apoptotic mechanism of cell death.
Assuntos
Doença de Lafora/genética , Proteínas Tirosina Fosfatases/genética , Animais , Ataxia/fisiopatologia , Comportamento Animal , Morte Celular , Fosfatases de Especificidade Dupla , Eletroencefalografia , Epilepsias Mioclônicas/fisiopatologia , Feminino , Corpos de Inclusão/metabolismo , Doença de Lafora/patologia , Doença de Lafora/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Sítio-Dirigida , Neurônios/metabolismo , Neurônios/ultraestrutura , Proteínas Tirosina Fosfatases/deficiência , Proteínas Tirosina Fosfatases/fisiologia , Proteínas Tirosina Fosfatases não Receptoras , Ubiquitina/metabolismoRESUMO
Los pacientes con enfermedad de Parkinson idiopático presentan pérdida neuronal de la sustancia negra parte compacta y la consecuente degeneración de la vía nigroestriatal; por otro lado, se ha reportado que los animales viejos también tienen pérdida de dicha inervación. El objetivo del presente trabajo fue comparar los cambios neurodegenerativos en el neuropilo del núcleo caudado de pacientes parkinsónicos y de animales viejos, con el fin de comparar los cambios que sufre dicho núcleo en los dos procesos degenerativos. En el análisis ultraestructural se utilizaron fragmentos del núcleo caudado de pacientes con enfermedad de Parkinson (n= 4) y de ratas viejas de 36 meses de edad (n= 4), para lo cual se midieron 100 botones presinápticos por caudado y se observó la estructura postsináptica y el número de contactos que establecía el botón con la misma estructura. Los resultados muestran que tanto los pacientes con enfermedad de Parkinson como las ratas viejas presentan un incremento estadisticamente significativo en el tamaño de los botones sinápticos, aumento en el número de contactos axodendríticos y aumento en el número de contactos dobles en comparación con los controles. Los datos indican que los pacientes parkinsónicos presentan alteraciones ultraestructurales similares a las de los animales viejos, alteraciones debidas a la ineficiente inervación dopaminérgica procedente del mesencéfalo, por lo cual se considero que el envejecimiento en animales es un buen modelo para el estudio de la neurodegeneración.