OBJECTIVE: To analyze the dynamic variant and clinical subtype of a pedigree affected with spinocerebellar ataxia (SCA) by using fluorescent-labeled primer combined with capillary electrophoresis. METHODS: Genomic DNA was extracted from 8 members including 6 patients and 2 healthy individuals from the pedigree. Six pairs of fluorescent-labeled primers were designed to screen pathological variants in association with common subtypes of SCA including SCA1, SCA2, SCA3, SCA6, SCA12 and SCA17.The PCR products were detected by capillary electrophoresis. RESULTS: The number of CAG repeats in the SCA3 gene of the proband were determined as 8 and 70, exceeded the normal range(12 to 40), which suggested a diagnosis of SCA3. The other five patients were all detected with abnormal CAG repeats in the SCA3 gene, while the two healthy individuals were determined to be within the normal range. CONCLUSION The abnormal expansion of CAG repeats in the SCA3 gene probably underlay the pathogenesis of the disease in this pedigree. Combined fluorescent-labeled primers PCR and capillary electrophoresis can detect dynamic variants among SCA patients with efficiency and accuracy.
Ataxin-3/genetics* ; Genetic Variation ; Humans ; Machado-Joseph Disease/genetics* ; Pedigree ; Repressor Proteins/genetics* ; Trinucleotide Repeats/genetics*

BACKGROUNDSpinocerebellar ataxias (SCAs) are a group of neurodegenerative disorders that primarily cause the degeneration in the cerebellum, spinal cord, and brainstem. We study the clinical characteristics, radiological features and gene mutation in Chinese families with SCAs.

METHODSIn this study, we investigated 10 SCAs Chinese families with SCA1, SCA3/Machado-Joseph disease (MJD), SCA7, SCA8. There were 27 people who were genetically diagnosed as SCA, of which 21 people showed clinical symptoms, and 6 people had no clinical phenotype that we called them presymptomatic patients. In addition, 3 people with cerebellar ataxia and cataracts were diagnosed according to the Harding diagnostic criteria but failed to be recognized as SCAs on genetic testing. Clinical characteristic analyses of each type of SCAs and radiological examinations were performed.

RESULTSWe found that SCA3/MJD was the most common subtype in Han population in China, and the ratio of the pontine tegmentum and the posterior fossa area was negatively correlated with the number of cytosine-adenine-guanine (CAG) repeats; the disease duration was positively correlated with the International Cooperative Ataxia Rating Scale score; and the CAG repeats number of abnormal alleles was negatively correlated with the age of onset.

CONCLUSIONSCollectively our study is a systematic research on SCAs in China, which may help for the clinical diagnosis and prenatal screening of this disease, and it may also aid toward better understanding of this disease.

Adult ; DNA Repeat Expansion ; genetics ; Female ; Humans ; Machado-Joseph Disease ; genetics ; pathology ; Male ; Mutation ; genetics ; Spinocerebellar Ataxias ; genetics ; pathology ; Trinucleotide Repeat Expansion ; genetics

OBJECTIVETo investigate the role of autophagy on the pathogenesis of spinocerebellar ataxia 3/Machado-Joseph disease (SCA3/MJD).

METHODSHEK293 cells expressing polyglutamine-expanded ataxin-3 were used as cell model for SCA3/MJD. The level of polyglutamine-expanded ataxin-3 was detected after cells were treated with different inhibitors or inducer of autophagy.

RESULTSInhibition of autophagy increased aggregate formation and cell death in HEK293 cells expressing mutated ataxin-3, and vice versa.

CONCLUSIONThe data suggested that autophagy is involved in the degradation of mutant ataxin-3, resulting in a decrease in the proportions of aggregate-containing cells and cell death in HEK293 cells expressing polyglutamine-expanded ataxin-3. It is possible that autophagy may be applied as a potential therapeutic approach for SCA3/MJD.

Ataxin-3 ; Autophagy ; Cell Line ; Humans ; Machado-Joseph Disease ; genetics ; metabolism ; physiopathology ; Mutation ; Nerve Tissue Proteins ; genetics ; metabolism ; Nuclear Proteins ; genetics ; metabolism ; Peptides ; metabolism ; Repressor Proteins ; genetics ; metabolism

To date, nearly 28 distinct genetic loci of autosomal dominant cerebellar ataxias have been identified, among them 18 disease-causing genes have been cloned. Of these, Machado-Joseph disease (MJD), also named as spinocerebellar ataxia type 3 (SCA3), is perhaps the most common subtype among different races and origins in the world. It is a neurodegenerative disease caused by the expansion of a CAG repeat in the coding region of the MJD1 gene, with obvious clinical and genetic heterogeneity. In this review, authors covered the recent advances in molecular genetic of SCA3/MJD.
Ataxin-3 ; Humans ; Machado-Joseph Disease ; genetics ; Molecular Biology ; Mutation ; Nerve Tissue Proteins ; chemistry ; genetics ; metabolism ; Nuclear Proteins ; chemistry ; genetics ; metabolism ; Repressor Proteins ; chemistry ; genetics ; metabolism

OBJECTIVEMachado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is a dominant neurodegenerative disorder caused by an expansion of the polyglutamine (polyQ) tract in MJD-1 gene product, ataxin-3 (AT3). This disease is characterized by the formation of intraneuronal inclusions, but the mechanism underlying their formation is still poorly understood. The present study is to explore the relationship between wild type (WT) AT3 and polyQ expanded AT3.

METHODSMouse neuroblastoma (N2a) cells or HEK293 cells were co-transfected with WT AT3 and different truncated forms of expanded AT3. The expressions of WT AT3 and the truncated forms of expanded AT3 were detected by Western blotting, and observed by an inverted fluorescent microscope. The interactions between AT3 and different truncated forms of expanded AT3 were detected by immunoprecipitation and GST pull-down assays.

RESULTSUsing fluorescent microscope, we observed that the truncated forms of expanded AT3 aggregate in transfected cells, and the full-length WT AT3 is recruited onto the aggregates. However, no aggregates were observed in cells transfected with the truncated forms of WT AT3. Immunoprecipitation and GST pull-down analyses indicate that WT AT3 interacts with the truncated AT3 in a polyQ length-dependent manner.

CONCLUSIONWT AT3 deposits in the aggregation that was formed by polyQ expanded AT3, which suggests that the formation of AT3 aggregation may affect the normal function of WT AT3 and increase polyQ protein toxicity in MJD.

Animals ; Ataxin-3 ; Blotting, Western ; Cell Line ; Immunoprecipitation ; Machado-Joseph Disease ; metabolism ; Mice ; Microscopy, Fluorescence ; Nuclear Proteins ; genetics ; metabolism ; Peptides ; metabolism ; Transcription Factors ; genetics ; metabolism ; Transfection

Polyglutamine (polyQ) diseases are a group of hereditary neurodegenerative disorders caused by expansion of a glutamine repeat in responsible gene products. To date, the pathogenesis of polyQ diseases is still not very clear, but many researches suggest that phosphorylation of mutant proteins plays a critical role on the process of Huntington's disease, dentatorubral-pallidoluysian atrophy, spinal bulbar muscular atrophy, spinocerebellar ataxia1 and spinocerebellar ataxia 3/Machado-Joseph disease.
Heredodegenerative Disorders, Nervous System ; genetics ; metabolism ; Humans ; Huntington Disease ; genetics ; metabolism ; Machado-Joseph Disease ; genetics ; metabolism ; Muscular Atrophy, Spinal ; Peptides ; genetics ; metabolism ; Phosphorylation ; physiology ; Spinocerebellar Degenerations ; genetics ; metabolism ; Trinucleotide Repeat Expansion ; genetics ; physiology ; Trinucleotide Repeats ; genetics

OBJECTIVETo explore the subcellular localization of ataxin-3 and the effect of polyglutamine (polyQ) expansion mutation on the morphology of mitochondrion, golgi apparatus and endoplasmic reticulum.

METHODSTransient transfection was employed to build cell models expressing wild-type or mutant ataxin-3 proteins. Indirect immunofluorescence was applied to identify markers of organelle membrane. The results were observed under a laser scanning confocal microscope.

RESULTSNo co-localization was observed for ataxin-3 protein and mitochondrial marker TOM20, but the percentage of cells with mitochondrial fragmentation has increased in cells expressing mutant ataxin-3 (P<0.05). No co-localization was observed for ataxin-3 protein and golgi marker GM130, and mutant ataxin-3 did not cause golgi fragmentation. Wide type and polyQ-expanded ataxin-3 both showed partial co-localization with ER marker calnexin. The latter showed more overlap with calnexin, and the overlapping signals were mostly located in the places where aggregates were situated.

CONCLUSIONPolyQ-expanded ataxin-3 protein may indirectly affect the integrity of mitochondria, but may cause no effect on the structure and functions of golgi apparatus. Endoplasmic reticulum may be another place where extended ataxin-3 protein can induce cytotoxicity in addition to the nucleus.

Ataxin-3 ; Cytoplasm ; genetics ; metabolism ; Endoplasmic Reticulum ; genetics ; metabolism ; HeLa Cells ; Humans ; Machado-Joseph Disease ; genetics ; metabolism ; Mitochondria ; genetics ; metabolism ; Nerve Tissue Proteins ; genetics ; metabolism ; Nuclear Proteins ; genetics ; metabolism ; Protein Transport ; Repressor Proteins ; genetics ; metabolism

OBJECTIVETo study the clinical features and gene mutation analysis in Machado-Joseph disease of spinocerebellar ataxia type 3 in littoral of Zhejiang.

METHODSClinical manifestation and brain MRI data 18 patients with SCA in family were analyzed. The gene mutations of 18 patients and 10 family numbers without abnormal presentation, and 12 healthy persons of controls.

RESULTSThe gene mutations of 18 patients is SCA3/MJD, and 2 asymptomatic SCA3/MJD had been detected in SCA family. Normal alleles of SCA3/MJD have CAG repeats ranging from 14 to 27, patients from 67 to 82, asymptomatic and carrier SCA3/MJD from 28 to 45. The main features of 18 patients included gait ataxia, ambiguity in speech and action clumsiness. Brain MRI showed remarkable atrophy on cerebellum and brain stem.

CONCLUSIONCAG expansions were related to SCA3/MJD. The clinical manifestations are ataxia and dysarthria. The detection of repeated times CAG can provide an effective way for the genetic and asymptomatic diagnosis.

Adolescent ; Adult ; Ataxin-3 ; Brain ; diagnostic imaging ; China ; Female ; Humans ; Machado-Joseph Disease ; diagnosis ; diagnostic imaging ; genetics ; Male ; Middle Aged ; Mutation ; Nerve Tissue Proteins ; genetics ; Nuclear Proteins ; genetics ; Pedigree ; Radiography ; Repressor Proteins ; genetics ; Trinucleotide Repeat Expansion ; Young Adult

OBJECTIVETo establish a stable, accurate and intuitive method for detecting the CAG trinucleotide repeats of MJD1 gene.

METHODSThe CAG trinucleotide polymorphism of the MJD1 gene was analyzed by recombinant DNA technology and DNA sequencing in 35 spinocerebellar ataxia 3/Machado-Joseph disease (SCA3/MJD) patients from Mainland China.

RESULTSThe range of the CAG repeat of the 35 patients was 65-81 (mean = 72.96 +/- 4.24). The CAG repeats contained two CAAs and one AAG variations in the CAG motif in all the patients and majority of the healthy controls. There was a CGG/GGG polymorphism at the 3' end of the CAG repeat. The GGG allele was consistently associated with smaller CAG repeats in healthy controls. On the other hand, the CGG allele consistently existed in the patients.

CONCLUSIONRecombinant DNA technology can stably, accurately and intuitively detect the CAG trinucleotide repeat of the MJD1 gene. It should be used as a major technique to diagnose the SCA3/MJD and analyze the polymorphism of CAG sequence.

Adolescent ; Adult ; Ataxin-3 ; Base Sequence ; Female ; Genetic Engineering ; methods ; Humans ; Machado-Joseph Disease ; genetics ; Male ; Middle Aged ; Molecular Sequence Data ; Nerve Tissue Proteins ; genetics ; Nuclear Proteins ; genetics ; Polymorphism, Genetic ; Repressor Proteins ; genetics ; Sequence Analysis, DNA ; Trinucleotide Repeats ; Young Adult

OBJECTIVETo study the possible relationship between the mitochondrial DNA (mtDNA) mutation and spinocerebellar ataxia type 3 (SCA3).

METHODSGenetic diagnosis of SCA3 was made by detecting the CAG-repeat expansion of MJD1 gene using PCR and DNA sequencing techniques. Then polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was performed to analyze the mitochondrial DNA extracted from peripheral white blood cells from 43 patients and presymptomatic individuals diagnosed according to CAG expansion, and 30 healthy individuals. Mitochondrial DNAs of subjects with abnormal SSCP were sequenced.

RESULTSA new mitochondrial DNA deletion of 9 bp at mtDNA 8282-8290 was identified in 1 patient and 3 presymptomatic individuals.

CONCLUSIONA new deletion mutation of mitochondrial DNA in 1 SCA3 patient and 3 presymptomatic individuals is reported.

Adolescent ; Adult ; Aged ; Base Sequence ; Case-Control Studies ; Child ; Child, Preschool ; DNA Mutational Analysis ; DNA, Mitochondrial ; genetics ; Female ; Humans ; Machado-Joseph Disease ; genetics ; Male ; Middle Aged ; Molecular Sequence Data ; Mutation ; Polymerase Chain Reaction ; Polymorphism, Single-Stranded Conformational ; Sequence Deletion