Adult ; Ataxin-3 ; genetics ; Female ; Humans ; Male ; Middle Aged ; Pedigree ; Repressor Proteins ; genetics ; Spinocerebellar Ataxias ; genetics

OBJECTIVE: To determine the frequency of different subtypes of spinocerebellar ataxias (SCAs) in the Han nationality of Hunan province in China. METHODS: The mutations of SCA1, SCA2, SCA3, SCA6, SCA7, SCA17, and dentatorulral-pallidoluysian (DRPLA) were detected with the polymerase chain reaction (PCR), denaturing polyacrylamide gel and DNA sequencing techniques in 139 autosomal dominant SCA families and 61 sporadic SCA patients. RESULTS: Of the 139 families, 11 (7.9%) were positive for SCA1, 9(6.5%) were positive for SCA2, 71 (51.1%) were positive for SCA3, 4 (2.9%) were positive for SCA6, 2 (1.4%) were positive for SCA7, and none was positive for SCA17 and DRPLA. There was 1 SCA2 patient, 3 SCA3 patients, 1 SCA6 patient in the 61 sporadic SCA patients. CONCLUSION The frequency of SCA3 is substantially higher than that of SCA1 and SCA2 in the autosomal dominant SCA patients in the Han nationality of Hunan province. SCA6 and SCA7 are rare subtypes.
Adolescent ; Adult ; Ataxin-1 ; Ataxin-3 ; Ataxin-7 ; Ataxins ; Child ; China ; ethnology ; DNA Mutational Analysis ; Female ; Humans ; Male ; Middle Aged ; Nerve Tissue Proteins ; genetics ; Nuclear Proteins ; genetics ; Repressor Proteins ; genetics ; Spinocerebellar Ataxias ; classification ; diagnosis ; genetics ; Trinucleotide Repeats ; genetics

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*

OBJECTIVEMachado-Joseph disease (MJD)/Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disorder caused by an expansion of polyglutamine tract near the C-terminus of the MJD1 gene product, ataxin-3. The precise mechanism of the MJD/SCA3 pathogenesis remains unclear. A growing body of evidence demonstrates that phosphorylation plays an important role in the pathogenesis of many neurodegenerative diseases. However, few kinases are known to phosphorylate ataxin-3. The present study is to explore whether ataxin-3 is a substrate of casein kinase 2 (CK2).

METHODSThe interaction between ataxin-3 and CK2 was identified by glutathione S-transferase (GST) pull-down assay and co-immunoprecipition assay. The phosphorylation of ataxin-3 by CK2 was measured by in vitro phosphorylation assays. Results (1) Both wild type and expanded ataxin-3 interacted with CK2alpha and CK2beta in vitro. (2) In 293 cells, both wild type and expanded ataxin-3 interacted with CK2beta, but not CK2alpha. (3) CK2 phosphorylated wild type and expanded ataxin-3.

CONCLUSIONAtaxin-3 is a substrate of protein kinase CK2.

Ataxin-3 ; Casein Kinase II ; metabolism ; Cell Line, Transformed ; Glutathione Transferase ; metabolism ; Humans ; Immunoprecipitation ; methods ; Nerve Tissue Proteins ; metabolism ; Nuclear Proteins ; metabolism ; Phosphorylation ; Repressor Proteins ; metabolism ; Transfection ; methods

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

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

OBJECTIVETo investigate the normal range of (CAG)n in spinocerebellar ataxia type 1 (SCA1) gene and spinocerebellar ataxia type 3 (SCA3/MJD) gene in 110 normal subjects of Han population in Northeastern China, to assess the genotypes for clinically diagnosed spinocerebellar ataxia(SCA) individuals including 25 patients from 8 families and 6 sporadic patients, and to make presymptomatic and prenatal diagnosis.

METHODSDNA fragments from the normal subjects and the patients were detected by fluorescence-PCR. Homozygosities were selected for DNA sequencing.

RESULTSThe normal ranges of (CAG)n of SCA1 and SCA3/MJD were 20-39 and 14-38 repeats respectively, SCA1 was found mostly to be 26 and 27 repeats, allele frequency 34.09% and 20.91%; heterozygosity was 84.55%, SCA3/MJD was found mostly to be 14 repeats, allele frequency 39.55%, heterozygosity was 78.18%.(CAG)(68) of SCA3/MJD gene of one affected individual had been found in a family but no CAG mutative expansion in related members was observed.

CONCLUSIONThe normal ranges of CAG repeats vary with areas and races. SCAs genotyping is the first choice in presymptomatic and prenatal diagnosis.

Ataxin-1 ; Ataxin-3 ; Ataxins ; China ; DNA ; chemistry ; genetics ; Family Health ; Female ; Gene Frequency ; Genotype ; Humans ; Machado-Joseph Disease ; diagnosis ; genetics ; Male ; Nerve Tissue Proteins ; genetics ; Nuclear Proteins ; genetics ; Pedigree ; Repressor Proteins ; Sequence Analysis, DNA ; Spinocerebellar Ataxias ; diagnosis ; genetics ; Trinucleotide Repeat Expansion ; genetics ; Trinucleotide Repeats ; genetics

OBJECTIVE: To construct the eukaryotic expression vector of MJD1 with normal copies of CAG trinucleotide repetition and MJD1 with CAG trinucleotide repetition expansion mutation respectively, and to determine whether the polyglutamine expansion in ataxin-3 could lead to the formation of intranuclear aggregation. METHODS: The coding sequence of wild-type MJD1 and mutant MJD1 was amplified by PCR from pAS2-1-MJD20Q and pAS2-1-MJD68Q respectively. After being digested with BamH I and Hind III, the PCR products were inserted into pcDNA3. 1-Myc-His(-) B. The recombinant plasmids pcDNA3.1-Myc-His(-) B-MJD20Q and pcDNA3.1-Myc-His(-) B-MJD68Q were identified by enzyme digestion analysis and DNA sequencing. The recombinant plasmid was transfected into SH-SYSY cells and the expression of MJD1 in the transfected cells was analyzed by Western blot. The immunofluorescence of the transfected cells was examined using a confocal microscope to observe the formation of intranuclear aggregation. RESULTS: Enzyme digestion analysis and DNA sequencing showed that the target gene was cloned into pcDNA3. 1-Myc-His(-) B. The expression of MJD1 in the transfected cells was confirmed by Western blot; The SH-SY5Y cells transfected with pcDNA3. 1-Myc-His(-) B-MJD68Q showed the formation of intranuclear aggregation, but the cells transfected with pcDNA3.1-Myc-His(-) B-MJD20Q did not show such phenomenon. CONCLUSION The eukaryotic expression vectors of MJD1 has been successfully constructed; The polyglutamine expansion in ataxin-3 could lead to the formation of intranuclear aggregation.
Ataxin-1 ; Ataxin-3 ; Ataxins ; Base Sequence ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; Humans ; Mediator Complex ; Molecular Sequence Data ; Nerve Tissue Proteins ; biosynthesis ; genetics ; Neuroblastoma ; metabolism ; pathology ; Nuclear Proteins ; biosynthesis ; genetics ; Plasmids ; genetics ; Receptors, Thyroid Hormone ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Repressor Proteins ; biosynthesis ; genetics ; Transfection ; Tumor Cells, Cultured

OBJECTIVETo clone an A3IP gene and investigate its cellular and histological localization based on previous research which has identified part of A3IP sequence interacting with carboxyl-terminal of ataxin-3.

METHODSBioinformatic and Northern blotting were applied to clone the A3IP gene and detect the expression of its transcripts in various human tissues and brain regions. Western blotting and immunofluorescence staining were applied to detect expression of A3IP protein in cultured cells. Immunohistochemistry staining was applied to study the expression of A3IP protein in various human tissues and brain regions.

RESULTScDNA cloning of A3IP gene's reading frame and its sequence assembly were completed. Three transcripts (1 kb, 1.35 kb and 6 kb, respectively) of A3IP were found to express in various human tissues and brain regions. A3IP pEGFP expresses in cytoplasm of cultured COS-7 cells and various human tissues and brain regions including cerebral cortex, cerebellum, muscle, peripheral nerve, liver and kidney.

CONCLUSIONThe cloned A3IP gene encodes A3IP, a novel ataxin-3 interacting protein. Three transcripts of A3IP are expressed in various human tissues and brain regions. A3IP is a cytosolic protein.

Ataxin-3 ; Base Sequence ; Carrier Proteins ; genetics ; metabolism ; Cloning, Molecular ; Humans ; Molecular Sequence Data ; Nerve Tissue Proteins ; genetics ; metabolism ; Nuclear Proteins ; genetics ; metabolism ; Protein Binding ; Protein Transport ; Repressor Proteins ; genetics ; metabolism