RESUMEN
Premature stop codons in CACNA1A, which encodes the alpha(1A) subunit of neuronal P/Q-type (Ca(V)2.1) Ca(2+) channels, cause episodic ataxia type 2 (EA2). CACNA1A undergoes extensive alternative splicing, which contributes to the pharmacological and kinetic heterogeneity of Ca(V)2.1-mediated Ca(2+) currents. We identified three novel heterozygous stop codon mutations associated with EA2 in an alternately spliced exon (37A), which encodes part of an EF-hand motif required for Ca(2+)-dependent facilitation. One family had a C to G transversion (Y1854X). A dinucleotide deletion results in the same premature stop codon in a second family, and a further single nucleotide change leads to a different truncation (R1858X) in a de novo case of EA2. Expression studies of the Y1854X mutation revealed loss of Ca(V)2.1-mediated current. Because these mutations do not affect the alternate exon 37B, these findings reveal unexpected dependence of cerebellar function on intact exon 37A-containing Ca(V)2.1 channels.
Asunto(s)
Empalme Alternativo/genética , Canales de Calcio Tipo N/genética , Codón sin Sentido/genética , Motivos EF Hand/genética , Mutación del Sistema de Lectura/genética , Isoformas de Proteínas/genética , Ataxias Espinocerebelosas/genética , Adulto , Animales , Cerebelo/química , Cerebelo/fisiopatología , Codón sin Sentido/química , Exones/genética , Femenino , Variación Genética/genética , Humanos , Masculino , Linaje , Isoformas de Proteínas/química , Ataxias Espinocerebelosas/diagnóstico , Ataxias Espinocerebelosas/fisiopatología , Xenopus laevisRESUMEN
We have established that the frequency of LRRK2 mutations in a series of 118 cases of familial Parkinson's disease is 5.1%. In the largest family with autosomal dominant, late-onset Parkinson's disease where affected subjects share a Y1699C missense mutation we provide a detailed clinical, pathological and imaging report. The phenotype in this large British kindred included asymmetrical, levodopa-responsive parkinsonism where unilateral leg tremor at onset and foot dystonia were prominent features. There was no significant abnormality of cognition but there was prominent behavioural disorder. We observed a lower age of onset in successive generations. Histopathology in one patient showed substantia nigra cell loss and Lewy body formation, with small numbers of cortical Lewy bodies. 18F-dopa positron emission tomography (PET) in another patient showed a pattern of nigrostriatal dysfunction typical of idiopathic Parkinson's disease. 18F-dopa-PET scans in unaffected family members prior to identifying the disease locus did not detect subclinical nigrostriatal dysfunction. Olfaction was assessed in affected subjects and Lewy bodies were identified in the olfactory bulb as well as cortex and brainstem of one deceased patient. In order to assess the role of mutations in this gene in other familial cases we undertook a mutation screen of all 51 exons of LRRK2 in 117 other smaller British kindreds with familial Parkinson's disease. The commonest mutation was G2019S and we also identified two novel mutations, R1941H and T2356I, in the coding sequence. These data suggest that parkinsonism caused by mutations in LRRK2 is likely to represent the commonest locus for autosomal dominant Parkinson's disease with a phenotype, pathology and in vivo imaging similar to idiopathic, late-onset Parkinson's disease.
Asunto(s)
Mutación Missense , Enfermedad de Parkinson/genética , Proteínas Serina-Treonina Quinasas/genética , Adulto , Edad de Inicio , Anciano , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Análisis Mutacional de ADN , Inglaterra , Femenino , Genes Dominantes , Ligamiento Genético , Pruebas Genéticas , Haplotipos , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Escala de Lod , Masculino , Persona de Mediana Edad , Mutación Missense/genética , Bulbo Olfatorio/patología , Enfermedad de Parkinson/diagnóstico , Enfermedad de Parkinson/patología , Linaje , Tomografía de Emisión de Positrones , RadiofármacosRESUMEN
The molecular basis of idiopathic generalized epilepsy remains poorly understood. Absence epilepsy with 3 Hz spike-wave EEG is one of the most common human epilepsies, and is associated with significant morbidity. Several spontaneously occurring genetic mouse models of absence epilepsy are caused by dysfunction of the P/Q-type voltage-gated calcium channel CaV2.1. Such mice exhibit a primary generalized spike-wave EEG, with frequencies in the range of 5-7 Hz, often associated with ataxia, evidence of cerebellar degeneration and abnormal posturing. Previously, we identified a single case of severe primary generalized epilepsy with ataxia associated with CaV2.1 dysfunction, suggesting a possible link between this channel and human absence epilepsy. We now report a family in which absence epilepsy segregates in an autosomal dominant fashion through three generations. Five members exhibit a combination of absence epilepsy (with 3 Hz spike-wave) and cerebellar ataxia. In patients with the absence epilepsy/ataxia phenotype, genetic marker analysis was consistent with linkage to the CACNA1A gene on chromosome 19, which encodes the main pore-forming alpha1A subunit of CaV2.1 channels (CaV2.1alpha1). DNA sequence analysis identified a novel point mutation resulting in a radical amino acid substitution (E147K) in CaV2.1alpha1, which segregated with the epilepsy/ataxia phenotype. Functional expression studies using human CACNA1A cDNA demonstrated that the E147K mutation results in impairment of calcium channel function. Impaired function of the brain calcium channel CaV2.1 may have a central role in the pathogenesis of certain cases of primary generalized epilepsy, particularly when associated with ataxia, which may be wrongly ascribed to anticonvulsant medication.
Asunto(s)
Canales de Calcio Tipo N/fisiología , Ataxia Cerebelosa/genética , Epilepsia Tipo Ausencia/genética , Adulto , Animales , Canales de Calcio/genética , Ataxia Cerebelosa/complicaciones , Ataxia Cerebelosa/fisiopatología , Electrofisiología , Epilepsia Tipo Ausencia/complicaciones , Epilepsia Tipo Ausencia/fisiopatología , Femenino , Genes Dominantes , Ligamiento Genético , Humanos , Masculino , Persona de Mediana Edad , Oocitos/metabolismo , Linaje , Mutación Puntual , Análisis de Secuencia de ADN/métodos , Xenopus laevisRESUMEN
Episodic ataxia type 2 (EA2) is an autosomal dominant channelopathy characterized by paroxysmal cerebellar ataxia. Previous studies suggest that most EA2 cases are associated with mutations in the alpha1A subunit of the P/Q-type voltage-gated calcium channel gene CACNA1A. In a UK national study, the authors analyzed 15 index cases with typical EA2 and identified two unreported intronic mutations that predict aberrant splicing.
Asunto(s)
Empalme Alternativo/genética , Canales de Calcio/genética , Ataxia Cerebelosa/genética , Mutación/genética , ARN/genética , Adolescente , Adulto , Ataxia Cerebelosa/metabolismo , Ataxia Cerebelosa/fisiopatología , Niño , Preescolar , Mapeo Cromosómico , Cromosomas Humanos Par 19/genética , Análisis Mutacional de ADN , Femenino , Genes Dominantes , Marcadores Genéticos/genética , Predisposición Genética a la Enfermedad/genética , Pruebas Genéticas , Humanos , Lactante , Intrones/genética , Masculino , Linaje , Polimorfismo Genético/genética , Subunidades de Proteína/genética , Sitios de Empalme de ARN/genética , Reino UnidoRESUMEN
Mutations of KCNA1, which codes for the K(+) channel subunit hKv1.1, are associated with the human autosomal dominant disease episodic ataxia type 1 (EA1). Five recently described mutations are associated with a broad range of phenotypes: neuromyotonia alone or with seizures, EA1 with seizures, or very drug-resistant EA1. Here we investigated the consequences of each mutation for channel assembly, trafficking, gating and permeation. We related data obtained from co-expression of mutant and wild-type hKv1.1 to the results of expressing mutant-wild-type fusion proteins, and combined electrophysiological recordings in Xenopus oocytes with a pharmacological discrimination of the contribution of mutant and wild-type subunits to channels expressed at the membrane. We also applied confocal laser scanning microscopy to measure the level of expression of either wild-type or mutant subunits tagged with green fluorescent protein (GFP). R417stop truncates most of the C-terminus and is associated with severe drug-resistant EA1. Electrophysiological and pharmacological measurements indicated that the mutation impairs both tetramerisation of R417stop with wild-type subunits, and membrane targeting of heterotetramers. This conclusion was supported by confocal laser scanning imaging of enhanced GFP (EGFP)-tagged hKv1.1 subunits. Co-expression of R417stop with wild-type hKv1.2 subunits yielded similar results to co-expression with wild-type hKv1.1. Mutations associated with typical EA1 (V404I) or with neuromyotonia alone (P244H) significantly affected neither tetramerisation nor trafficking, and only altered channel kinetics. Two other mutations associated with a severe phenotype (T226R, A242P) yielded an intermediate result. The phenotypic variability of KCNA1 mutations is reflected in a wide range of disorders of channel assembly, trafficking and kinetics.