Myasthenia gravis coexisting with HINT1-related motor axonal neuropathy without neuromyotonia: a case report.

BACKGROUND: HINT1 mutations cause an autosomal recessive axonal neuropathy with neuromyotonia. This is a first case report of coexistence of myasthenia gravis (MG) and HINT1-related motor axonal neuropathy without neuromyotonia. CASE PRESENTATION: A 32-year-old woman presented with recurrent ptosis for 8 years, diplopia for 2 years and limb weakness for 1 year and a half. Neostigmine test, elevated AChR antibody level and positive repetitive nerve stimulation supported the diagnosis of MG. Electroneurography (ENG) and electromyography (EMG) examinations revealed a motor axonal neuropathy without neuromyotonic or myokymic discharges. Next-generation sequencing and Sanger sequencing were performed to identify the gene responsible for suspected hereditary neuropathy. Genetic testing for a HINT1 mutation was performed and revealed a homozygous mutation at c.278G>T (p. G93V). The patient was treated with pyridostigmine, oral prednisolone and azathioprine. Her ptosis and diplopia have significantly improved at 6-month follow-up. CONCLUSIONS: Concurrence of MG and hereditary motor axonal neuropathy without neuromyotonia is quite rare. Detection of ptosis with or without ophthalmoplegia, distribution of limb weakness, and reflex can help in recognizing the combination of MG and peripheral neuropathy. Early diagnosis is important for initial treatment and prognosis. The novel homozygous variant c.278G>T(p.G93V) contributes to the pathogenic variants spectrum of the HINT1 gene.

Preliminary Study of hsa-mir-626 Change in the Cerebrospinal Fluid in Parkinson's Disease.

CONTEXT: A host of microRNAs have been reported to suppress tumor growth, invasion, and metastasis and play roles in neurodegeneration disorders. Moreover, microRNA changes are found in the peripheral blood, cerebrospinal fluid (CSF), and brain tissues of central nervous system diseases, including glioma, Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis, and depression. Compared with other body fluids, CSF can reflect the brain pathological processes more accurately. AIMS: To understand whether microRNA expression may be misregulated in patients with PD, and further discover potential diagnostic biomarkers and promising therapeutic targets for PD. MATERIALS AND METHODS: Here, through real-time reverse-transcription polymerase chain reaction (RT-PCR), we compared CSF microRNA from 15 PD patients, 11 AD patients, and 16 controls with other neurologic disorders, such as encephalitis and Guillain-Barre syndrome. RESULTS: Finally, we identified hsa-miR-626 changes in the CSF of PD patients. The mean expression level of hsa-miR-626 was significantly reduced in the CSF of PD patients compared with AD patients and controls. CONCLUSIONS: Our approach provides a preliminary research for identifying biomarkers in the CSF that could be used for the detection, diagnosis, and monitoring of PD.

Preliminary study of hsa-miR-626 change in the cerebrospinal fluid of Parkinson's disease patients.

microRNAs have been reported to suppress tumor growth, invasion, and metastasis and play roles in neurodegeneration disorders. Moreover, changes in microRNAs are found in the peripheral blood, cerebrospinal fluid (CSF), and brain tissues in patients of central nervous system diseases, including glioma, Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis and depression. Compared with other bodily fluids, CSF is the most accurate at representing the pathological processes of the brain. To understand whether microRNA expression may be dysregulated in the patients of PD, and to further discover potential diagnostic biomarkers and promising therapeutic targets for PD, we used real-time polymerase chain reaction (RT-PCR) to compare CSF microRNAs from 20 PD patients, 13 AD patients and 27 controls with other neurologic disorders such as encephalitis and Guillain-Barre syndrome. Finally, we found that the mean expression level of hsa-miR-626 was significantly reduced in the CSF of patients with PD compared with AD and controls. Our approach potentially identified a biomarker in CSF that upon further investigation, could be used for the detection, diagnosis, and monitoring of PD in combination with other PD biomarkers.

SUMO-1 modification on K166 of polyQ-expanded ataxin-3 strengthens its stability and increases its cytotoxicity.

Post-translational modification by SUMO was proposed to modulate the pathogenesis of several neurodegenerative diseases. Spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) is an autosomal dominant neurodegenerative disease caused by polyQ-expanded ataxin-3. We have previously shown that ataxin-3 was a new target of SUMOylation in vitro and in vivo. Here we identified that the major SUMO-1 binding site was located on lysine 166. SUMOylation did not influence the subcellular localization, ubiquitination or aggregates formation of mutant-type ataxin-3, but partially increased its stability and the cell apoptosis. Our findings revealed the role of ataxin-3 SUMOylation in SCA3/MJD pathogenesis.

[The advances in research on phosphorylation of polyglutamine disease].

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.

[Sequence polymorphism of the promoter region of gene STK11 in patients with Peutz-Jeghers syndrome].

OBJECTIVE: To explore the relationship between the sequence variation of the promoter region (-1543 approximately -1160) of STK11 gene and the risk of developing Peutz-Jeghers syndrome (PJS). METHODS: The sequences of the promoter region of 14 PJS patients (7 patients are inherited and the other 7 patients are sporadic) and 42 normal individuals were PCR amplified and then sequenced. RESULTS: A new single nucleotide polymorphism (SNP) G/T (-1275) in STK11 promoter region was identified. The frequency of genotype GG, GT, and TT was 53.3%, 26.7%, and 20%, respectively among PJS patients and 33.3%, 64.3%, and 2.4%, respectively among the normal individuals. The frequency of genotype GG and TT among patients was significantly higher than that among the normal individuals, and the frequency of genotype GT among patients was significantly lower than that among the normal individuals (chi(2)=8.521, P<0.05). CONCLUSION: G/T(-1275) in STK11 promoter region is a new SNP. The genotype of this new SNP may relate to the risk of developing Peutz-Jeghers syndrome (PJS) deserve further research.

[Clinical, pathological and genetic studies in a Chinese Charcot-Marie-Tooth disease type 2 family].

OBJECTIVE: To report a Chinese Charcot-Marie-Tooth disease type 2 (CMT2) family. METHODS: All the members in the family were studied clinically,and 6 patients were studied electrophysiologically. Sural nerve biopsy was performed in the proband. PMP22 gene duplications were detected by highly polymorphic short tandem repeat. Point mutation analysis of PMP22, MPZ and NEFL gene was screened by PCR-SSCP combined with DNA direct sequencing. A genome-wide screening was carried out to the family. RESULT: Except 2 who had weakness and atrophy in both proximal and distal muscles of the lower limbs, all patients presented muscle wasting and a predominating weakness of distal parts of the lower limbs, and mild to moderate sensory impairments. In 6 patients who were subjected to elctrophysiological examinations, median-nerve conduction velocity (NCV) of the median nerve was normal. Electromyograms (EMGs) revealed signs of denervation with large motor unit potentials, fibrillation potentials and positive sharp waves. Sural nerve biopsy of the proband confirmed the presence of axonal neuropathy with an important loss of large myelinating fibers and a large number of clusters with mostly thinly myelinated axons. PMP22, MPZ and NEFL gene mutations were not found. The results of genome-wide screening revealed a linkage of CMT2 to a locus at chromosome 12q24. CONCLUSION: The results are consistent with the diagnosis of CMT2. This family represents a rare genetic type of CMT2 which can be designated as CMT2L.

[Research on clinical characteristics and ATM gene mutations in Chinese patients with ataxia-telangiectasia].

OBJECTIVE: To investigate the clinical manifestation and mutation characteristics of ATM gene in Chinese patients with ataxia-telangiectasia (AT). METHODS: Sequence variants of the entire coding exons of ATM gene were tested using polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), polyacrylamide gel electrophoresis (PAGE) and DNA direct sequencing in two Chinese patients clinically diagnosed as AT. RESULTS: The clinical characteristics of two AT patients were progressive cerebellar ataxia with ages at onset of childhood, ocular-cutaneous telangiectasia and recurrent pulmonary infection due to immuno-deficiency; the serum alpha fetoprotein (AFP) levels were higher than normal, the serum immunoglobin IgA and IgG levels were lower than normal; brain MRI showed cerebellar atrophy, brain SPECT showed cerebellar regional cerebral blood flow (rCBF) hypoperfusion to a certain degree. Totally three nucleotide changes were identified. A missense mutation of G1346C in exon 11, which was a homozygotic mutation, was identified in one patient; a nonsense mutation of G610T in exon 6 combined with a missense mutation of C6679T in exon 47, which was a compound heterozygotic mutation, were identified in the other patient. They were co-segregated with the disease and were localized within the functional domain of ATM gene. CONCLUSION: We have made gene diagnoses for two Chinese AT patients, in which three novel ATM gene mutations were identified.

Research on screening and identification of proteins interacting with ataxin-3.

OBJECTIVE: 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). METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Construction of the eukaryotic expression vector of MJD1 and its expression in SH-SY5Y cells].

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.