RESUMO
CAG repeat expansion is the genetic cause of nine incurable polyglutamine (polyQ) diseases with neurodegenerative features. Silencing repeat RNA holds great therapeutic value. Here, we developed a repeat-based RNA-cleaving DNAzyme that catalyzes the destruction of expanded CAG repeat RNA of six polyQ diseases with high potency. DNAzyme preferentially cleaved the expanded allele in spinocerebellar ataxia type 1 (SCA1) cells. While cleavage was non-allele-specific for spinocerebellar ataxia type 3 (SCA3) cells, treatment of DNAzyme leads to improved cell viability without affecting mitochondrial metabolism or p62-dependent aggresome formation. DNAzyme appears to be stable in mouse brain for at least 1 month, and an intermediate dosage of DNAzyme in a SCA3 mouse model leads to a significant reduction of high molecular weight ATXN3 proteins. Our data suggest that DNAzyme is an effective RNA silencing molecule for potential treatment of multiple polyQ diseases.
Assuntos
DNA Catalítico/administração & dosagem , DNA Catalítico/genética , Doença de Machado-Joseph/genética , Peptídeos/genética , RNA/genética , Expansão das Repetições de Trinucleotídeos/genética , Animais , Ataxina-3/genética , Linhagem Celular Tumoral , Inativação Gênica/fisiologia , Células HEK293 , Humanos , Doença de Machado-Joseph/terapia , Camundongos , Peptídeos/metabolismo , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/terapia , Técnicas EstereotáxicasRESUMO
Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disease caused by CAG (encoding glutamine) repeat expansion in the Ataxin-3 (ATXN3) gene. We have shown previously that ATXN3-depleted or pathogenic ATXN3-expressing cells abrogate polynucleotide kinase 3'-phosphatase (PNKP) activity. Here, we report that ATXN3 associates with RNA polymerase II (RNAP II) and the classical nonhomologous end-joining (C-NHEJ) proteins, including PNKP, along with nascent RNAs under physiological conditions. Notably, ATXN3 depletion significantly decreased global transcription, repair of transcribed genes, and error-free double-strand break repair of a 3'-phosphate-containing terminally gapped, linearized reporter plasmid. The missing sequence at the terminal break site was restored in the recircularized plasmid in control cells by using the endogenous homologous transcript as a template, indicating ATXN3's role in PNKP-mediated error-free C-NHEJ. Furthermore, brain extracts from SCA3 patients and mice show significantly lower PNKP activity, elevated p53BP1 level, more abundant strand-breaks in the transcribed genes, and degradation of RNAP II relative to controls. A similar RNAP II degradation is also evident in mutant ATXN3-expressing Drosophila larval brains and eyes. Importantly, SCA3 phenotype in Drosophila was completely amenable to PNKP complementation. Hence, salvaging PNKP's activity can be a promising therapeutic strategy for SCA3.
Assuntos
Ataxina-3/genética , Reparo do DNA por Junção de Extremidades , Enzimas Reparadoras do DNA/metabolismo , Doença de Machado-Joseph/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , RNA Polimerase II/metabolismo , Proteínas Repressoras/genética , Idoso de 80 Anos ou mais , Animais , Animais Geneticamente Modificados , Ataxina-3/metabolismo , Encéfalo/patologia , Linhagem Celular , Quebras de DNA de Cadeia Dupla , Modelos Animais de Doenças , Drosophila , Feminino , Técnicas de Silenciamento de Genes , Humanos , Células-Tronco Pluripotentes Induzidas , Doença de Machado-Joseph/metabolismo , Doença de Machado-Joseph/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação , Peptídeos/genética , RNA Interferente Pequeno/metabolismoRESUMO
Although the main clinical manifestations of spinocerebellar ataxias (SCAs) result from damage of the cerebellum, other systems may also be involved. Olfactory deficits have been reported in other types of ataxias, especially in SCA3; however, there are no studies on olfactory deficits in SCA type 10 (SCA10). To analyze olfactory function of SCA10 patients compared with that of SCA3, Parkinson's, and healthy controls. Olfactory identification was tested in three groups of 30 patients (SCA10, SCA3, and Parkinson's disease (PD)) and 44 healthy controls using the Sniffin' Sticks (SS16) test. Mean SS16 score was 11.9 ± 2.9 for the SCA10 group, 12.3 ± 1.9 for the SCA3 group, 6.6 ± 2.8 for the PD group, and 12.1 ± 2.0 for the control group. Mean SS16 score for the SCA10 group was not significantly different from the scores for the SCA3 and control groups but was significantly higher than the score for the PD group (p < 0.001) when adjusted for age, gender, and history of smoking. There was no association between SS16 scores and disease duration in the SCA10 or SCA3 groups or number of repeat expansions. SS16 and Mini Mental State Examination scores were correlated in the three groups: SCA10 group (r = 0.59, p = 0.001), SCA3 group (r = 0.50, p = 0.005), and control group (r = 0.40, p = 0.007). We found no significant olfactory deficits in SCA10 in this large series.
Assuntos
Doença de Machado-Joseph/fisiopatologia , Transtornos do Olfato/fisiopatologia , Doença de Parkinson/fisiopatologia , Olfato , Ataxias Espinocerebelares/fisiopatologia , Expansão das Repetições de DNA/genética , Feminino , Humanos , Doença de Machado-Joseph/genética , Masculino , Pessoa de Meia-Idade , Transtornos do Olfato/genética , Estudos Prospectivos , Ataxias Espinocerebelares/genéticaRESUMO
Spinocerebellar ataxia type 10 (SCA10), an autosomal dominant cerebellar ataxia disorder, is caused by a non-coding ATTCT microsatellite repeat expansion in the ataxin 10 gene. In a subset of SCA10 families, the 5'-end of the repeat expansion contains a complex sequence of penta- and heptanucleotide interruption motifs which is followed by a pure tract of tandem ATCCT repeats of unknown length at its 3'-end. Intriguingly, expansions that carry these interruption motifs correlate with an epileptic seizure phenotype and are unstable despite the theory that interruptions are expected to stabilize expanded repeats. To examine the apparent contradiction of unstable, interruption-positive SCA10 expansion alleles and to determine whether the instability originates outside of the interrupted region, we sequenced approximately 1 kb of the 5'-end of SCA10 expansions using the ATCCT-PCR product in individuals across multiple generations from four SCA10 families. We found that the greatest instability within this region occurred in paternal transmissions of the allele in stretches of pure ATTCT motifs while the intervening interrupted sequences were stable. Overall, the ATCCT interruption changes by only one to three repeat units and therefore cannot account for the instability across the length of the disease allele. We conclude that the AT-rich interruptions locally stabilize the SCA10 expansion at the 5'-end but do not completely abolish instability across the entire span of the expansion. In addition, analysis of the interruption alleles across these families support a parsimonious single origin of the mutation with a shared distant ancestor.
Assuntos
Ataxina-10/genética , Epilepsia/genética , Padrões de Herança , Repetições de Microssatélites , Mutação , Ataxias Espinocerebelares/genética , Alelos , Sequência de Bases , Epilepsia/patologia , Feminino , Expressão Gênica , Variação Genética , Humanos , Masculino , Linhagem , Fenótipo , Ataxias Espinocerebelares/patologiaRESUMO
Myotonic dystrophy type 1 (DM1) is caused by expanded Cytosine-Thymine-Guanine (CTG) repeats in the 3'-untranslated region (3' UTR) of the Dystrophia myotonica protein kinase (DMPK) gene, for which there is no effective therapy. The objective of this study is to develop genome therapy in human DM1 induced pluripotent stem (iPS) cells to eliminate mutant transcripts and reverse the phenotypes for developing autologous stem cell therapy. The general approach involves targeted insertion of polyA signals (PASs) upstream of DMPK CTG repeats, which will lead to premature termination of transcription and elimination of toxic mutant transcripts. Insertion of PASs was mediated by homologous recombination triggered by site-specific transcription activator-like effector nuclease (TALEN)-induced double-strand break. We found genome-treated DM1 iPS cells continue to maintain pluripotency. The insertion of PASs led to elimination of mutant transcripts and complete disappearance of nuclear RNA foci and reversal of aberrant splicing in linear-differentiated neural stem cells, cardiomyocytes, and teratoma tissues. In conclusion, genome therapy by insertion of PASs upstream of the expanded DMPK CTG repeats prevented the production of toxic mutant transcripts and reversal of phenotypes in DM1 iPS cells and their progeny. These genetically-treated iPS cells will have broad clinical application in developing autologous stem cell therapy for DM1.
Assuntos
Terapia Genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Distrofia Miotônica/genética , Distrofia Miotônica/metabolismo , Transplante de Células-Tronco , Animais , Diferenciação Celular , Núcleo Celular/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Marcação de Genes , Loci Gênicos , Humanos , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Distrofia Miotônica/terapia , Miotonina Proteína Quinase/genética , Poli A , Ligação Proteica , Splicing de RNA , Teratoma/genética , Teratoma/metabolismo , Teratoma/patologia , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição , Transplante Autólogo , Repetições de TrinucleotídeosRESUMO
Spinocerebellar ataxia type 10 (SCA10) is a rare form of autosomal dominant ataxia found predominantly in patients from Latin America with Amerindian ancestry. The authors report the history of SCA10 families from the south of Brazil (the states of Paraná and Santa Catarina), emphasizing the Belgian-Amerindian connection.
A ataxia espinocerebellar tipo 10 (AEC10) é uma forma rara de ataxia cerebelar autossômica dominante, encontrada predominantemente em pacientes da América Latina, de origem Ameríndia. Os autores relatam a história de famílias com AEC10 do sul do Brasil (estados do Paraná e Santa Catarina), enfatizando a conexão Ameríndia-Belga.
Assuntos
Humanos , Indígenas Sul-Americanos/etnologia , Ataxias Espinocerebelares/etnologia , Bélgica/etnologia , Brasil/etnologia , Expansão das Repetições de DNA , Mapas como Assunto , FenótipoRESUMO
The authors review ataxia telangiectasia, emphasizing historical aspects, genetic discoveries, and the clinical presentations of the classical and atypical forms. In fact, ataxia telangiectasia represents a multisystem entity with pleomorphic neurological and systemic manifestations. ATM syndrome is proposed as a more adequate designation for this entity.
Assuntos
Ataxia Telangiectasia/diagnóstico , Ataxia Telangiectasia/história , Animais , Ataxia Telangiectasia/genética , História do Século XX , História do Século XXI , Humanos , Mutação , Proteínas Serina-Treonina Quinases/genética , PubMed/estatística & dados numéricosRESUMO
Myotonic dystrophy type 1 (DM1) is caused by expanded CTG repeats in the 3'-untranslated region (3' UTR) of the DMPK gene. Correcting the mutation in DM1 stem cells would be an important step toward autologous stem cell therapy. The objective of this study is to demonstrate in vitro genome editing to prevent production of toxic mutant transcripts and reverse phenotypes in DM1 stem cells. Genome editing was performed in DM1 neural stem cells (NSCs) derived from human DM1 induced pluripotent stem (iPS) cells. An editing cassette containing SV40/bGH polyA signals was integrated upstream of the CTG repeats by TALEN-mediated homologous recombination (HR). The expression of mutant CUG repeats transcript was monitored by nuclear RNA foci, the molecular hallmarks of DM1, using RNA fluorescence in situ hybridization. Alternative splicing of microtubule-associated protein tau (MAPT) and muscleblind-like (MBNL) proteins were analyzed to further monitor the phenotype reversal after genome modification. The cassette was successfully inserted into DMPK intron 9 and this genomic modification led to complete disappearance of nuclear RNA foci. MAPT and MBNL 1, 2 aberrant splicing in DM1 NSCs were reversed to normal pattern in genome-modified NSCs. Genome modification by integration of exogenous polyA signals upstream of the DMPK CTG repeat expansion prevents the production of toxic RNA and leads to phenotype reversal in human DM1 iPS-cells derived stem cells. Our data provide proof-of-principle evidence that genome modification may be used to generate genetically modified progenitor cells as a first step toward autologous cell transfer therapy for DM1.
Assuntos
Genoma Humano , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas Associadas aos Microtúbulos/metabolismo , Distrofia Miotônica/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Pluripotentes/citologia , Humanos , Distrofia Miotônica/patologia , FenótipoRESUMO
Next-generation sequencing was used to investigate 9 rare Chinese pedigrees with rare autosomal recessive neurologic Mendelian disorders. Five probands with ataxia-telangectasia and 1 proband with chorea-acanthocytosis were analyzed by targeted gene sequencing. Whole-exome sequencing was used to investigate 3 affected individuals with Joubert syndrome, nemaline myopathy, or spastic ataxia Charlevoix-Saguenay type. A list of known and novel candidate variants was identified for each causative gene. All variants were genetically verified by Sanger sequencing or quantitative polymerase chain reaction with the strategy of disease segregation in related pedigrees and healthy controls. The advantages of using next-generation sequencing to diagnose rare autosomal recessive neurologic Mendelian disorders characterized by genetic and phenotypic heterogeneity are demonstrated. A genetic diagnostic strategy combining the use of targeted gene sequencing and whole-exome sequencing with the aid of next-generation sequencing platforms has shown great promise for improving the diagnosis of neurologic Mendelian disorders.
Assuntos
Sequência de Bases/genética , Exoma/genética , Genes Recessivos/genética , Técnicas de Diagnóstico Molecular/métodos , Doenças do Sistema Nervoso/diagnóstico , Doenças do Sistema Nervoso/genética , Análise de Sequência de DNA/métodos , Anormalidades Múltiplas , Adulto , Povo Asiático/genética , Ataxia , Encéfalo/patologia , Doenças Cerebelares , Cerebelo/anormalidades , Criança , Pré-Escolar , Anormalidades do Olho , Feminino , Humanos , Lactente , Recém-Nascido , Deficiência Intelectual , Doenças Renais Císticas , Imageamento por Ressonância Magnética , Masculino , Espasticidade Muscular , Miopatias da Nemalina , Doenças do Sistema Nervoso/patologia , Neuroacantocitose , Atrofia Óptica , Retina/anormalidades , Ataxias Espinocerebelares , Telangiectasia , Adulto JovemRESUMO
Dystrophia myotonica type 1 (DM1) is an autosomal dominant multisystem disorder. The pathogenesis of central nervous system (CNS) involvement is poorly understood. Disease-specific induced pluripotent stem cell (iPSC) lines would provide an alternative model. In this study, we generated two DM1 lines and a normal iPSC line from dermal fibroblasts by retroviral transduction of Yamanaka's four factors (hOct4, hSox2, hKlf4, and hc-Myc). Both DM1 and control iPSC clones showed typical human embryonic stem cell (hESC) growth patterns with a high nuclear-to-cytoplasm ratio. The iPSC colonies maintained the same growth pattern through subsequent passages. All iPSC lines expressed stem cell markers and differentiated into cells derived from three embryonic germ layers. All iPSC lines underwent normal neural differentiation. Intranuclear RNA foci, a hallmark of DM1, were detected in DM1 iPSCs, neural stem cells (NSCs), and terminally differentiated neurons and astrocytes. In conclusion, we have successfully established disease-specific human DM1 iPSC lines, NSCs, and neuronal lineages with pathognomonic intranuclear RNA foci, which offer an unlimited cell resource for CNS mechanistic studies and a translational platform for therapeutic development.
Assuntos
Diferenciação Celular , Derme/metabolismo , Fibroblastos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Distrofia Miotônica/metabolismo , Células-Tronco Neurais/metabolismo , Linhagem Celular , Derme/patologia , Feminino , Fibroblastos/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Masculino , Pessoa de Meia-Idade , Distrofia Miotônica/patologia , Distrofia Miotônica/terapia , Células-Tronco Neurais/patologia , Transplante de Células-TroncoAssuntos
Adulto , Feminino , Humanos , Glucocorticoides/efeitos adversos , Metilprednisolona/efeitos adversos , Ataxias Espinocerebelares/induzido quimicamente , Doença Aguda , Expansão das Repetições de DNA/genética , Imageamento por Ressonância Magnética , Púrpura Trombocitopênica/tratamento farmacológico , Ataxias Espinocerebelares/genéticaRESUMO
Spinocerebellar ataxia type 2 (SCA2) is caused by triple nucleotide repeat (CAG) expansion in the coding region of the ATAXN2 gene on chromosome 12, which produces an elongated, toxic polyglutamine tract, leading to Purkinje cell loss. There is currently no effective therapy. One of the main obstacles that hampers therapeutic development is lack of an ideal disease model. In this study, we have generated and characterized SCA2-induced pluripotent stem (iPS) cell lines as an in vitro cell model. Dermal fibroblasts (FBs) were harvested from primary cultures of skin explants obtained from a SCA2 subject and a healthy subject. For reprogramming, hOct4, hSox2, hKlf4, and hc-Myc were transduced to passage-3 FBs by retroviral infection. Both SCA2 iPS and control iPS cells were successfully generated and showed typical stem cell growth patterns with normal karyotype. All iPS cell lines expressed stem cell markers and differentiated in vitro into cells from three embryonic germ layers. Upon in vitro neural differentiation, SCA2 iPS cells showed abnormality in neural rosette formation but successfully differentiated into neural stem cells (NSCs) and subsequent neural cells. SCA2 and normal FBs showed a comparable level of ataxin-2 expression; whereas SCA2 NSCs showed less ataxin-2 expression than normal NSCs and SCA2 FBs. Within the neural lineage, neurons had the most abundant expression of ataxin-2. Time-lapsed neural growth assay indicated terminally differentiated SCA2 neural cells were short-lived compared with control neural cells. The expanded CAG repeats of SCA2 were stable throughout reprogramming and neural differentiation. In conclusion, we have established the first disease-specific human SCA2 iPS cell line. These mutant iPS cells have the potential for neural differentiation. These differentiated neural cells harboring mutations are invaluable for the study of SCA2 pathogenesis and therapeutic drug development.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Proteínas do Tecido Nervoso/genética , Neurogênese , Ataxias Espinocerebelares/genética , Adulto , Ataxinas , Linhagem Celular Transformada , Células Cultivadas , Reprogramação Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Ataxias Espinocerebelares/patologia , TransfecçãoRESUMO
The main clinical manifestations of the spinocerebellar ataxias (SCAs) result from the involvement of the cerebellum and its connections. Cerebellar activity has been consistently observed in functional imaging studies of olfaction, but the anatomical pathways responsible for this connection have not yet been elucidated. Previous studies have demonstrated olfactory deficit in SCA2, Friedreich's ataxia and in small groups of ataxia of diverse aetiology. The authors used a validated version of the 16-item smell identification test from Sniffin' Sticks (SS-16) was used to evaluate 37 patients with genetically determined autosomal dominant ataxia, and 31 with familial ataxia of unknown genetic basis. This data was also compared with results in 106 Parkinson's disease patients and 218 healthy controls. The SS-16 score was significantly lower in ataxia than in the control group (p<0.001, 95% CI for ß=0.55 to 1.90) and significantly higher in ataxia than in Parkinson's disease (p<0.001, 95% CI for ß=-4.58 to -3.00) when adjusted for age (p=0.001, 95% CI for ß=-0.05 to -0.01), gender (p=0.19) and history of tobacco use (p=0.41). When adjusted for general cognitive function, no significant difference was found between the ataxia and control groups. This study confirms previous findings of mild hyposmia in ataxia, and further suggests this may be due to general cognitive deficits rather than specific olfactory problems.
Assuntos
Ataxia Cerebelar/complicações , Ataxia Cerebelar/fisiopatologia , Cerebelo/fisiopatologia , Disfunção Cognitiva/complicações , Transtornos do Olfato/etiologia , Percepção Olfatória , Doença de Parkinson/complicações , Adulto , Idoso , Brasil , Ataxia Cerebelar/genética , Disfunção Cognitiva/etiologia , Disfunção Cognitiva/fisiopatologia , Feminino , Testes Genéticos , Humanos , Masculino , Pessoa de Meia-Idade , Transtornos do Olfato/fisiopatologia , Doença de Parkinson/genética , Doença de Parkinson/fisiopatologia , Ataxias Espinocerebelares/complicações , Ataxias Espinocerebelares/fisiopatologiaRESUMO
Deciphering the contribution of genetic instability in somatic cells is critical to our understanding of many human disorders. Myotonic dystrophy type 1 (DM1) is one such disorder that is caused by the expansion of a CTG repeat that shows extremely high levels of somatic instability. This somatic instability has compromised attempts to measure intergenerational repeat dynamics and infer genotype-phenotype relationships. Using single-molecule PCR, we have characterized more than 17 000 de novo somatic mutations from a large cohort of DM1 patients. These data reveal that the estimated progenitor allele length is the major modifier of age of onset. We find no evidence for a threshold above which repeat length does not contribute toward age at onset, suggesting pathogenesis is not constrained to a simple molecular switch such as nuclear retention of the DMPK transcript or haploinsufficiency for DMPK and/or SIX5. Importantly, we also show that age at onset is further modified by the level of somatic instability; patients in whom the repeat expands more rapidly, develop the symptoms earlier. These data establish a primary role for somatic instability in DM1 severity, further highlighting it as a therapeutic target. In addition, we show that the level of instability is highly heritable, implying a role for individual-specific trans-acting genetic modifiers. Identifying these trans-acting genetic modifiers will facilitate the formulation of novel therapies that curtail the accumulation of somatic expansions and may provide clues to the role these factors play in the development of cancer, aging and inherited disease in the general population.
Assuntos
Distrofia Miotônica/etiologia , Distrofia Miotônica/genética , Característica Quantitativa Herdável , Expansão das Repetições de Trinucleotídeos , Idade de Início , Idoso , Alelos , Estudos de Associação Genética , Instabilidade Genômica , Haploinsuficiência/genética , Proteínas de Homeodomínio/genética , Humanos , Pessoa de Meia-Idade , Distrofia Miotônica/epidemiologia , Miotonina Proteína Quinase , Proteínas Serina-Treonina Quinases/genéticaRESUMO
OBJECTIVE: Spinocerebellar ataxias are neurodegenerative disorders involving the cerebellum and its connections. There are more than 30 distinct subtypes, 16 of which are associated with an identified gene. The aim of the current study was to evaluate a large group of patients from 104 Brazilian families with spinocerebellar ataxias. METHODS: We studied 150 patients from 104 families with spinocerebellar ataxias who had received molecular genetic testing for spinocerebellar ataxia types 1, 2, 3, 6, 7, 8, 10, 12, 17, and dentatorubral-pallidoluysian atrophy. A statistical analysis of the results was performed using basic descriptive statistics and the correlation coefficient (r), Student's t-test, chi-square test, and Yates' correction. The statistical significance level was established for p-values <0.05. RESULTS: The results show that the most common subtype was spinocerebellar ataxia 3, which was followed by spinocerebellar ataxia 10. Moreover, the comparison between patients with spinocerebellar ataxia 3, spinocerebellar ataxia 10, and other types of spinocerebellar ataxia revealed distinct clinical features for each type. In patients with spinocerebellar ataxia 3, the phenotype was highly pleomorphic, although the most common signs of disease included cerebellar ataxia (CA), ophthalmoplegia, diplopia, eyelid retraction, facial fasciculation, pyramidal signs, and peripheral neuropathy. In patients with spinocerebellar ataxia 10, the phenotype was also rather distinct and consisted of pure cerebellar ataxia and abnormal saccadic eye movement as well as ocular dysmetria. Patients with spinocerebellar ataxias 2 and 7 presented highly suggestive features of cerebellar ataxia, including slow saccadic ocular movements and areflexia in spinocerebellar ataxia 2 and visual loss in spinocerebellar ataxia 7. CONCLUSIONS: Spinocerebellar ataxia 3 was the most common subtype examined, followed by spinocerebellar ataxia 10. Patients with spinocerebellar ataxia 2 and 7 demonstrated highly suggestive features, whereas the phenotype of spinocerebellar ataxia 3 patients was highly pleomorphic and spinocerebellar ataxia 10 patients exhibited pure cerebellar ataxia. Epilepsy was absent in all of the patients with spinocerebellar ataxia 10 in this series.
Assuntos
Feminino , Humanos , Estudos de Associação Genética , Doença de Machado-Joseph/diagnóstico , Ataxias Espinocerebelares/diagnóstico , Idade de Início , Brasil , Expansão das Repetições de DNA/genética , Técnicas de Diagnóstico Molecular , Doença de Machado-Joseph/genética , Ataxias Espinocerebelares/genéticaRESUMO
Spinocerebellar ataxia 10 (SCA10) is an autosomal dominant disease caused by large-scale expansions of the (ATTCT)(n) repeat within an intron of the human ATXN10 gene. In contrast to other expandable repeats, this pentanucleotide repeat does not form stable intra- or interstranded DNA structures, being a DNA unwinding element instead. We analyzed the instability of the (ATTCT)(n) repeat in a yeast experimental system, where its expansions led to inactivation of the URA3 reporter gene. The inactivation was due to a dramatic decrease in the mRNA levels owing to premature transcription termination and RNA polyadenylation at the repeat. The rates of expansions strongly increased with the repeat's length, mimicking genetic anticipation in human pedigrees. A first round of genetic analysis showed that a functional TOF1 gene precludes, whereas a functional RAD5 gene promotes, expansions of the (ATTCT)(n) repeat. We hypothesize that repeat expansions could occur upon fortuitous template switching during DNA replication. The rate of repeat contractions was elevated in the Tof1 knockout strain, but it was not affected by the RAD5 gene. Supporting the notion of replication irregularities, we found that (ATTCT)(n) repeats also cause length-dependent chromosomal fragility in yeast. Repeat-mediated fragility was also affected by the Tof1 and Rad5 proteins, being reduced in their absence.
Assuntos
Expansão das Repetições de DNA/genética , DNA/metabolismo , Repetições de Microssatélites/genética , Proteínas do Tecido Nervoso/genética , Ataxina-10 , Sequência de Bases , Brasil , Clonagem Molecular , DNA/genética , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Inativação Gênica , Genes Reporter/genética , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Transformação GenéticaRESUMO
Myotonia congenita is a muscular disease characterized by myotonia, hypertrophy, and stiffness. It is inherited as either autosomal dominant or recessive known as Thomsen and Becker diseases, respectively. Here we confirm the clinical diagnosis of a family diagnosed with a myotonic condition many years ago and report a new mutation in the CLCN1 gene. The clinical diagnosis was established using ocular, cardiac, neurological and electrophysiological tests and the molecular diagnosis was done by PCR, SSCP and sequencing of the CLCN1 gene. The proband and the other affected individuals exhibited proximal and distal muscle weakness but no hypertrophy or muscular pain was found. The myotatic reflexes were lessened and sensibility was normal. Electrical and clinical myotonia was found only in the sufferers. Slit lamp and electrocardiogram tests were normal. Two affected probands presented diminution of the sensitive conduction velocities and prolonged sensory distal latencies. The clinical spectrum for this family is in agreement with a clinical diagnosis of Becker myotonia. This was confirmed by molecular diagnosis where a new disease-causing mutation (Q412P) was found in the family and absent in 200 unaffected chromosomes. No latent myotonia was found in this family; therefore the ability to cause this subclinical sign might be intrinsic to each mutation. Implications of the structure-function-genotype relationship for this and other mutations are discussed. Adequate clinical diagnosis of a neuromuscular disorder would allow focusing the molecular studies toward the confirmation of the initial diagnosis, leading to a proper clinical management, genetic counseling and improving in the quality of life of the patients and relatives.
La miotonía congénita es una enfermedad muscular caracterizada por miotonía, hipertrofia y rigidez. Se presenta con dos patrones de herencia, autosómica dominante en cuyo caso recibe el nombre de miotonía de Thomsen, o autosómica recesiva conocida como miotonía de Becker. En este trabajo se confirmó el diagnóstico clínico presuntivo hecho hace algunos años en una familia con una condición miotónica y se reporta una nueva mutación en el gen CLCN1. El diagnóstico clínico se estableció después de estudios oculares, cardíacos, neurológicos y electrofisiológicos. El diagnóstico molecular fue hecho mediante la PCR, SSCP y secuenciación del gen CLCN1. El caso índice y los otros individuos afectados exhibieron debilidad muscular proximal y distal, pero no se encontró hipertrofia ni dolor muscular. Los reflejos miotáticos estuvieron disminuidos y la sensibilidad fue normal. Se encontró miotonía clínica y eléctrica solo en los individuos afectados. Las pruebas de lámpara de hendidura y electrocardiograma resultaron normales. Dos individuos afectados presentaron disminución de las velocidades de conducción sensitiva y latencias distales sensoriales prolongadas. El cuadro clínico concuerda con la miotonía de Becker, lo cual se confirmó con el hallazgo de una mutación responsable de la enfermedad en el gen CLCN1 (Q412P), la cual se encontró en la familia y estuvo ausente en 200 cromosomas provenientes de la población general. No se encontró miotonía latente, por lo que probablemente la habilidad de causar este signo subclínico es intrínsica de cada mutación. Afinar el diagnóstico clínico diferencial de las enfermedades neuromusculares permitiría enfocar los estudios moleculares hacia la confirmación del diagnóstico inicial en forma eficiente, lo cual permitiría un manejo clínico y asesoramiento genético más adecuados y una mejora en la calidad de vida de los pacientes y sus familias.
Assuntos
Humanos , Masculino , Feminino , Criança , Adolescente , Adulto , Canais de Cloreto/genética , Miotonia Congênita/diagnóstico , Mutação/genética , Costa Rica , Enzimas de Restrição do DNA , Fenótipo , Linhagem , Marcadores Genéticos , Miotonia Congênita/genética , Polimorfismo Conformacional de Fita Simples , Reação em Cadeia da PolimeraseRESUMO
The authors report the history of spinocerebellar ataxia 10 (SCA10), since its first report in a large Portuguese-ancestry Family with autosomal dominant pure cerebellar ataxia, till the final identification of further families without Mexican ancestry. These families present a quite different phenotype from those SCA10 families described in Mexico.
Os autores apresentam a história da descoberta da ataxia espinocerebelar tipo 10 (AEC10) no Brasil, desde o primeiro relato em uma família com ancestrais portugueses com ataxia cerebelar pura, autossômica dominante, até a identificação de famílias sem ancestrais mexicanos. Essas famílias apresentam um fenótipo de AEC10, com ataxia cerebelar "pura", distinta daquele descrito nas famílias no México.