RESUMEN
Myotonic dystrophy (DM) is caused by either an untranslated CTG expansion in the 3' untranslated region of the DMPK gene on chromosome 19 (dystrophia myotonica type 1 [DM1]), or an untranslated CCTG tetranucleotide repeat expansion in intron 1 of the ZNF9 gene on chromosome 3 (dystrophia myotonica type 2 [DM2]). RNA-binding proteins adhere to transcripts of the repeat expansions that accumulate in the nucleus, and a trans-dominant dysregulation of pre-mRNA alternative splicing has been demonstrated for several genes. In muscle from patients with DM1, altered insulin-receptor splicing to the nonmuscle isoform corresponds to the insulin insensitivity and diabetes that are part of the DM phenotype; because of insulin-receptor species differences, this effect is not seen in mouse models of the disease. We now demonstrate that comparable splicing abnormalities occur in DM2 muscle prior to the development of muscle histopathology, thus demonstrating an early pathogenic effect of RNA expansions.
Asunto(s)
Empalme Alternativo/genética , Resistencia a la Insulina/genética , Distrofia Miotónica/genética , Receptor de Insulina/genética , Adulto , Anciano , Estudios de Casos y Controles , Femenino , Glucosa/metabolismo , Humanos , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Distrofia Miotónica/diagnóstico , Distrofia Miotónica/patología , Sondas ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Myotonic dystrophy (DM), the most common form of muscular dystrophy in adults, can be caused by a mutation on either chromosome 19q13 (DM1) or 3q21 (DM2/PROMM). DM1 is caused by a CTG expansion in the 3' untranslated region of the dystrophia myotonica-protein kinase gene (DMPK). Several mechanisms have been invoked to explain how this mutation, which does not alter the protein-coding portion of a gene, causes the specific constellation of clinical features characteristic of DM. We now report that DM2 is caused by a CCTG expansion (mean approximately 5000 repeats) located in intron 1 of the zinc finger protein 9 (ZNF9) gene. Parallels between these mutations indicate that microsatellite expansions in RNA can be pathogenic and cause the multisystemic features of DM1 and DM2.
Asunto(s)
Proteínas de Unión al ADN/genética , Intrones , Repeticiones de Microsatélite , Distrofia Miotónica/genética , Proteínas de Unión al ARN/genética , Dedos de Zinc , Alelos , Southern Blotting , Mapeo Cromosómico , Cromosomas Humanos Par 3/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Enfermedades en Gemelos/genética , Femenino , Humanos , Hibridación Fluorescente in Situ , Desequilibrio de Ligamiento , Escala de Lod , Masculino , Músculos/metabolismo , Mutación , Distrofia Miotónica/metabolismo , Fenotipo , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Gemelos Monocigóticos , Dedos de Zinc/genéticaRESUMEN
OBJECTIVE: To compare the clinical and genetic features of the seven-generation family (MN-A) used to define the spinocerebellar ataxia 8 (SCA8) locus. BACKGROUND: The authors recently described an untranslated CTG expansion that causes a novel form of SCA (SCA8) characterized by reduced penetrance and complex patterns of repeat instability. METHODS: Clinical and molecular features of 82 members of the MN-A family were evaluated by neurologic examination, quantitative dexterity testing, and, in some individuals, MRI and sperm analyses. RESULTS: SCA8 is a slowly progressive, predominantly cerebellar ataxia with marked cerebellar atrophy, affecting gait, swallowing, speech, and limb and eye movements. CTG tracts are longer in affected (mean = 116 CTG repeats) than in unaffected expansion carriers (mean = 90, p < 10-8). Quantitative dexterity testing did not detect even subtle signs of ataxia in unaffected expansion carriers. Surprisingly, all 21 affected MN-A family members inherited an expansion from their mothers. The maternal penetrance bias is consistent with maternal repeat expansions yielding alleles above the pathogenic threshold in the family (>107 CTG) and paternal contractions resulting in shorter alleles. Consistent with the reduced penetrance of paternal transmissions, CTG tracts in all or nearly all sperm (84 to 99) are significantly shorter than in the blood (116) of an affected man. CONCLUSIONS: The biologic relationship between repeat length and ataxia indicates that the CTG repeat is directly involved in SCA8 pathogenesis. Diagnostic testing and genetic counseling are complicated by the reduced penetrance, which often makes the inheritance appear recessive or sporadic, and by interfamilial differences in the length of a stable (CTA)n tract preceding the CTG repeat.
Asunto(s)
Cromosomas Humanos Par 13/genética , Ataxias Espinocerebelosas/genética , Adulto , Anciano , Encéfalo/patología , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Linaje , Ataxias Espinocerebelosas/patologíaRESUMEN
We recently described an untranslated CTG expansion that causes a previously undescribed form of spinocerebellar ataxia (SCA8). The SCA8 CTG repeat is preceded by a polymorphic but stable CTA tract, with the configuration (CTA)(1-21)(CTG)(n). The CTG portion of the repeat is elongated on pathogenic alleles, which nearly always change in size when transmitted from generation to generation. To better understand the reduced penetrance and maternal penetrance bias associated with SCA8 we analyzed the sequence configurations and instability patterns of the CTG repeat in affected and unaffected family members. In contrast to other triplet repeat diseases, expanded alleles found in affected SCA8 individuals can have either a pure uninterrupted CTG repeat tract or an allele with one or more CCG, CTA, CTC, CCA or CTT interruptions. Surprisingly, we found six different sequence configurations of the CTG repeat on expanded alleles in a seven generation family. In two instances duplication of CCG interruptions occurred over a single generation and in other instances duplications that had occurred in different branches of the family could be inferred. We also evaluated SCA8 instability in sperm samples from individuals with expansions ranging in size from 80 to 800 repeats in blood. Surprisingly the SCA8 repeat tract in sperm underwent contractions, with nearly all of the resulting expanded alleles having repeat lengths of <100 CTGs, a size that is not often associated with disease. These en masse repeat contractions in sperm likely underlie the reduced penetrance associated with paternal transmission.
Asunto(s)
Proteínas del Tejido Nervioso/genética , Espermatozoides/metabolismo , Repeticiones de Trinucleótidos/genética , Alelos , Southern Blotting , Salud de la Familia , Padre , Femenino , Duplicación de Gen , Humanos , Masculino , Modelos Genéticos , Madres , Proteínas del Tejido Nervioso/biosíntesis , Linaje , Penetrancia , ARN Largo no Codificante , ARN no Traducido , Análisis de Secuencia de ADN , Ataxias Espinocerebelosas/genética , Expansión de Repetición de TrinucleótidoRESUMEN
Spinocerebellar ataxia type 8 (SCA8) is a neurodegenerative disorder caused by the expansion of a CTG trinucleotide repeat that is transcribed as part of an untranslated RNA. As a step towards understanding the molecular pathology of SCA8, we have defined the genomic organization of the SCA8 RNA transcripts and assembled a 166 kb segment of genomic sequence containing the repeat. The most striking feature of the SCA8 transcripts is that the most 5' exon is transcribed through the first exon of another gene that is transcribed in the opposite orientation. This gene arrangement suggests that the SCA8 transcript is an endogenous antisense RNA that overlaps the transcription and translation start sites as well as the first splice donor sequence of the sense gene. The sense transcript encodes a 748 amino acid protein with a predicted domain structure typical of a family of actin-organizing proteins related to the Drosophila Kelch gene, and so has been given the name Kelch-like 1 (KLHL1). We have identified the full-length cDNA sequence for both the human and mouse KLHLI genes, and have elucidated the general genomic organization of the human gene. The predicted open reading frame and promoter region are highly conserved, and both genes are primarily expressed in specific brain tissues, including the cerebellum, the tissue most affected by SCA8. Transfection studies with epitope-tagged KLHL1 demonstrate that the protein localizes to the cytoplasm, suggesting that it may play a role in organizing the actin cytoskeleton of the brain cells in which it is expressed.
Asunto(s)
Encéfalo/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , ARN sin Sentido , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Northern Blotting , Células COS , Proteínas Portadoras/química , Proteínas Portadoras/genética , Secuencia Conservada , Citoplasma/metabolismo , ADN Complementario/metabolismo , Exones , Humanos , Intrones , Ratones , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Plásmidos , Regiones Promotoras Genéticas , Homología de Secuencia de Aminoácido , Ataxias Espinocerebelosas/genética , Distribución TisularRESUMEN
An individual with late-onset ataxia was found to be heterozygous for an unusual (GAAGGA)65 sequence and a normal GAA repeat in the frataxin gene. No frataxin point mutation was present, excluding a form of Friedreich ataxia. (GAAGGA)65 did not have the inhibitory effect on gene expression in transfected cells shown by pathogenic GAA repeats of similar length. GAA repeats, but not (GAAGGA)65, adopt a triple helical conformation in vitro. We suggest that such a triplex structure is essential for suppression of gene expression.
Asunto(s)
Ataxia de Friedreich/genética , Proteínas de Unión a Hierro , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Anciano , Alelos , Secuencia de Bases/genética , Humanos , Masculino , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , FrataxinaRESUMEN
Myotonic dystrophy (DM) is the only disease reported to be caused by a CTG expansion. We now report that a non-coding CTG expansion causes a novel form of spinocerebellar ataxia (SCA8). This expansion, located on chromosome 13q21, was isolated directly from the genomic DNA of an ataxia patient by RAPID cloning. SCA8 patients have expansions similar in size (107-127 CTG repeats) to those found among adult-onset DM patients. SCA8 is the first example of a dominant SCA not caused by a CAG expansion translated as a polyglutamine tract.
Asunto(s)
Degeneraciones Espinocerebelosas/genética , Repeticiones de Trinucleótidos , Regiones no Traducidas , Alelos , Femenino , Genes Dominantes , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Reacción en Cadena de la Polimerasa , Técnica del ADN Polimorfo Amplificado Aleatorio , Transcripción GenéticaRESUMEN
OBJECTIVE: To determine the incidence of spinocerebellar ataxia (SCA) types 1, 2, 3, 6, and 7 and Friedreich's ataxia (FA) among a large panel of ataxia families. BACKGROUND: The ataxias are a clinically and genetically heterogeneous group of neurodegenerative diseases that variably affect the cerebellum, brainstem, and spinocerebellar tracts. Trinucleotide repeat expansions have been shown to be the mutational mechanism for five dominantly inherited SCAs as well as FA. METHODS: We collected DNA samples and clinical data from patients representing 361 families with adult-onset ataxia of unknown etiology. Patients with a clinical diagnosis of FA were specifically excluded from our collection. RESULTS: Among the 178 dominant kindreds, we found SCA1 expansion at a frequency of 5.6%, SCA2 expansion at a frequency of 15.2%, SCA3 expansion at a frequency of 20.8%, SCA6 expansion at a frequency of 15.2%, and SCA7 expansion at a frequency of 4.5%. FA alleles were found in 11.4% of apparently recessive and 5.2% of apparently sporadic patients. Among these patients the repeat sizes for one or both FA alleles were relatively small, with sizes for the smaller allele ranging from 90 to 600 GAA repeats. The clinical presentation for these patients is atypical for FA, with one or more of the following characteristics: adult onset of disease, retained tendon reflexes, normal plantar response, and intact or partially intact sensory perceptions. CONCLUSIONS: Pathogenic trinucleotide repeat expansions were found among 61% of the dominant kindreds. Among patients with apparently recessive or negative family histories of ataxia, 6.8% and 4.4% tested positive for a CAG expansion at one of the dominant loci, and 11.4 and 5.2% of patients with apparently recessive or sporadic forms of ataxia had FA expansions. Because of the significant implications that a dominant versus recessive inheritance pattern has for future generations, it is important to screen patients who do not have a clearly dominant inheritance pattern for expansions at both the FA and the dominant ataxia loci.
Asunto(s)
Salud de la Familia , Ataxia de Friedreich/epidemiología , Ataxia de Friedreich/genética , Repeticiones de Trinucleótidos , Adulto , ADN/análisis , Genes Dominantes , Genes Recesivos , Homocigoto , Humanos , Incidencia , Persona de Mediana EdadRESUMEN
Trinucleotide repeat expansions have been shown to cause a number of neurodegenerative diseases. A hallmark of most of these diseases is the presence of anticipation, a decrease in the age at onset in consecutive generations due to the tendency of the unstable trinucleotide repeat to lengthen when passed from one generation to the next. The involvement of trinucleotide repeat expansions in a number of other diseases--including familial spastic paraplegia, schizophrenia, bipolar affective disorder and spinocerebellar ataxia type 7 (SCA7; ref. 10)--is suggested both by the presence of anticipation and by repeat expansion detection (RED) analysis of genomic DNA samples. The involvement of trinucleotide expansions in these diseases, however, can be conclusively confirmed only by the isolation of the expansions present in these populations and detailed analysis to assess each expansion as a possible pathogenic mutation. We describe a novel procedure for quick isolation of expanded trinucleotide repeats and the corresponding flanking nucleotide sequence directly from small amounts of genomic DNA by a process of Repeat Analysis, Pooled Isolation and Detection of individual clones containing expanded trinucleotide repeats (RAPID cloning). We have used this technique to clone the pathogenic SCA7 CAG expansion from an archived DNA sample of an individual affected with ataxia and retinal degeneration.