Detection and analysis of dynamic variant in a pedigree affected with spinocerebellar ataxia type 3 / 中华医学遗传学杂志

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.

Lithium carbonate and coenzyme Q10 reduce cell death in a cell model of Machado-Joseph disease

Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disorder caused by expansion of the polyglutamine domain of the ataxin-3 (ATX3) protein. MJD/SCA3 is the most frequent autosomal dominant ataxia in many countries. The mechanism underlying MJD/SCA3 is thought to be mainly related to protein misfolding and aggregation leading to neuronal dysfunction followed by cell death. Currently, there are no effective treatments for patients with MJD/SCA3. Here, we report on the potential use of lithium carbonate and coenzyme Q10 to reduce cell death caused by the expanded ATX3 in cell culture. Cell viability and apoptosis were evaluated by MTT assay and by flow cytometry after staining with annexin V-FITC/propidium iodide. Treatment with lithium carbonate and coenzyme Q10 led to a significant increase in viability of cells expressing expanded ATX3 (Q84). In addition, we found that the increase in cell viability resulted from a significant reduction in the proportion of apoptotic cells. Furthermore, there was a significant change in the expanded ATX3 monomer/aggregate ratio after lithium carbonate and coenzyme Q10 treatment, with an increase in the monomer fraction and decrease in aggregates. The safety and tolerance of both drugs are well established; thus, our results indicate that lithium carbonate and coenzyme Q10 are good candidates for further in vivo therapeutic trials.

Subcellular localization of ataxin-3 and its effect on the morphology of cytoplasmic organoids / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Transient 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.</p><p><b>RESULTS</b>No 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.</p><p><b>CONCLUSION</b>PolyQ-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.</p>

Cloning and localization of A3IP -a novel protein that interacts with ataxin-3 / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Bioinformatic 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.</p><p><b>RESULTS</b>cDNA 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.</p><p><b>CONCLUSION</b>The 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.</p>

Polyglutamine-expanded ataxin-3 is degraded by autophagy / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the role of autophagy on the pathogenesis of spinocerebellar ataxia 3/Machado-Joseph disease (SCA3/MJD).</p><p><b>METHODS</b>HEK293 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.</p><p><b>RESULTS</b>Inhibition of autophagy increased aggregate formation and cell death in HEK293 cells expressing mutated ataxin-3, and vice versa.</p><p><b>CONCLUSION</b>The 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.</p>

Identification of ATXN3 intermedial allele associated with a disease phenotype in an SCA3 Han Chinese family / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the clinical manifestation and the mutation characteristics of intermedial allele associated with a disease phenotype of a Machado-Joseph disease (MJD) family.</p><p><b>METHODS</b>Polymerase chain reaction, capillary electrophoresis, molecular cloning and sequencing were performed to detect the ATXN3 gene in an spinocerebellar ataxia(SCA) family. The fragments of expanded alleles were subcloned into the pGEM-T plasmids and sequenced.</p><p><b>RESULTS</b>The expanded repeats at the MJD locus were confirmed by molecular technique. The proband had 43 CAG repeats at the MJD locus. He had two sons with 41 and 64 repeats in the expanded allele respectively.</p><p><b>CONCLUSION</b>A 43 CAG repeat allele was unstable upon inter-generational transmission. The change of the CAG repeat was bidirectional. This is the shortest expanded allele associated with a disease phenotype in the MJD gene reported to date. The identification of the MJD family has reduced the amplitude between the normal and expanded allele repeats.</p>

Molecular and prenatal diagnosis of a pedigree with spinocerebellar ataxia / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify the type of a pedigree with spinocerebellar ataxia, and carry out asymptomatic carrier detection and prenatal diagnosis.</p><p><b>METHODS</b>The blood samples of two patients in the spinocerebellar ataxia pedigree were collected. Based on the clinical characteristics of the pedigree and the disease incidence in China, the regions containing the CAG repeat of the SCA1, SCA2 and SCA3/MJD genes were amplified by polymerase chain reaction (PCR). The numbers of CAG repeats in the normal and abnormal allele fragments were identified by using agarose gel electrophoresis and DNA sequencing. We further carried out tests on the children of the patients and fetus to identify the presence of the abnormal allele.</p><p><b>RESULTS</b>The numbers of CAG repeat in the SCA1 and SCA2 genes were in the normal range. The CAG repeat number in one allele of SCA3/MJD gene was in the normal range, while that in the other allele was in the abnormal range. One of the children of the patients and the fetus carried the abnormal allele.</p><p><b>CONCLUSION</b>It was confirmed that the pedigree was SCA3/MJD by gene diagnosis. One of the children of the patients was asymptomatic carrier and the fetus also carried the abnormal allele.</p>

Detection of the CAG trinucleotide repeats of MJD1 gene by recombinant DNA technology / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a stable, accurate and intuitive method for detecting the CAG trinucleotide repeats of MJD1 gene.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>Recombinant 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.</p>

Clinical features and gene mutation analysis in Machado-Joseph disease of spinocerebellar ataxia type 3 in littoral of Zhejiang / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the clinical features and gene mutation analysis in Machado-Joseph disease of spinocerebellar ataxia type 3 in littoral of Zhejiang.</p><p><b>METHODS</b>Clinical 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>CAG 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.</p>

PolyQ-expanded ataxin-3 interacts with full-length ataxin-3 in a polyQ length-dependent manner / 神经科学通报·英文版

<p><b>OBJECTIVE</b>Machado-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.</p><p><b>METHODS</b>Mouse 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.</p><p><b>RESULTS</b>Using 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.</p><p><b>CONCLUSION</b>WT 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.</p>

Casein kinase 2 interacts with and phosphorylates ataxin-3 / 神经科学通报·英文版

<p><b>OBJECTIVE</b>Machado-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).</p><p><b>METHODS</b>The 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.</p><p><b>CONCLUSION</b>Ataxin-3 is a substrate of protein kinase CK2.</p>

Recent advances in molecular genetics of spinocerebellar ataxia type 3/Machado-Joseph disease / 中华医学遗传学杂志

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.

Screening for proteins interacting with ataxin-3, the gene product of SCA3/MJD / 中南大学学报(医学版)

OBJECTIVE@#To screen for proteins interacting with ataxin-3 by yeast two-hybrid system 3, and to discuss the function of ataxin-3 and pathogenesis of spinocerebellar ataxia type 3 and Machado-Joseph disease (SCA3/MJD).@*METHODS@#First we sub-cloned the full reading frame of both wild-type and mutant ataxin-3 into carrier pGBKT7 (ataxin-3-bait), and then screened human brain cDNA library with ataxin-3-bait.@*RESULTS@#We found five positive clones in 6.5 x 10(6) transformers. After sequencing, we knew all of them were novel ataxin-3 interacting proteins. Three were corresponded to the known sequences coding the known proteins, which were human Rho GDP dissociation inhibitor alpha, small ubiquitin-like modifier 1, and human neuronal amiloride-sensitive cation channel 2. Another two of the five were unknown.@*CONCLUSION@#Small ubiquitin-like modifier 1 probably interacted with ataxin-3, suggesting that the sumoylation probably participated in post-translation modifying of ataxin-3 and pathogenesis of SCA3/MJD.

Frequency of different subtypes of spinocerebellar ataxia in the Han nationality of Hunan province in China / 中南大学学报(医学版)

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.

Construction of the eukaryotic expression vector of MJD1 and its expression in SH-SY5Y cells / 中南大学学报(医学版)

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.

Research on screening and identification of proteins interacting with ataxin-3 / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>This study sought to isolate and identify the proteins that interact with ataxin-3, to confirm the interacted domain, and to provide new clues for exploring the function of ataxin-3 and the pathogenesis of spinocerebellar ataxia type 3 and Machado-Joseph disease (SCA3/MJD).</p><p><b>METHODS</b>Yeast two-hybrid screen (MATCHMAKER GAL4 Two-Hybrid System 3) and regular molecular biologic techniques were undertaken to screen human brain cDNA library with mutant ataxin-3 bait. Two baits from both normal and mutant C-terminus of ataxin-3 were created by subcloned methods to determine which domain of ataxin-3 interacts with the putative associated proteins and to find out optimal candidate proteins that interact with C-terminus of ataxin-3. Confocal microscope was used to observe whether ataxin-3 co-localized with the obtained interacting proteins in mammalian cells.</p><p><b>RESULTS</b>Five novel ataxin-3 interacting proteins were obtained, among which were three known proteins, namely human rhodopsin guanosine diphosphate dissociation inhibitor alpha, small ubiquitin-like modifier 1, and human neuronal amiloride-sensitive cation channel 2; the other two were unknown. Interacting domain analysis revealed that an unknown protein interacted with the C-terminus near the polyglutamine tract of ataxin-3, the other four all interacted with the N-terminus. In the nucleus of SH-SY5Y cell, small ubiquitin-like modifier 1 co-localized with the wild-type ataxin-3 and with the intranuclear aggregates formed by the mutant ataxin-3.</p><p><b>CONCLUSION</b>An unknown protein probably interacting with C-terminus of ataxin-3 is firstly discovered, and the initiative findings suggest first that the interaction of small ubiquitin-like modifier 1 with N-terminus of ataxin-3 and the relevant sumoylation probably participate in the post-translation modifying of ataxin-3 and in the pathogenesis of SCA3/MJD.</p>

Analysis and application of SCA1 and SCA3/MJD gene CAG repeats in Han population in Northeastern China / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>DNA fragments from the normal subjects and the patients were detected by fluorescence-PCR. Homozygosities were selected for DNA sequencing.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>The normal ranges of CAG repeats vary with areas and races. SCAs genotyping is the first choice in presymptomatic and prenatal diagnosis.</p>