Expression and immunogenicity of hepatitis E virus-like particles based on recombinant truncated ORF2 capsid protein.

Hepatitis E is an emerging zoonotic disease, posing a severe threat to public health in the world. Since there are no specific treatments available for HEV infection, it is crucial to develop vaccine to prevent this infection. In this study, the truncated ORF2 encoded protein of 439aa∼617aa (HEV3-179) from HEV CCJD-517 isolates was expressed as VLPs in E. coli with diameters of approximate 20 nm. HEV3-179 protein was immunized with mice, and the results showed that a higher titre of antibody was induced in NIH mice in comparison with that of KM mice (P < 0.01) and BALB/c mice (P < 0.01). The induced antibody titer is much higher in subcutaneous immunization mice than that in the mice inoculated via abdominal immunization (P < 0.05) and muscles immunization (P < 0.01). Mice immunized with 12 µg and 6 µg candidate vaccine induced higher level of antibody titer than that of 3 µg dosage group (P < 0.01, P < 0.05). Antibody change curve showed that HEV IgG antibody titer increased from 14 days post immunization (dpi) to 1:262144 and reached the peak level on 42 dpi before gradually retreated with the same level antibody titer with 1:131072 until 84 dpi. Mice inoculated with HEV3-179 produced higher titer of cytokines than the mock group, and the concentration of IL-1ß (P < 0.01) and IFN-γ (P < 0.01) further increased after stimulated by candidate vaccine. The result indicated that HEV3-179 possesses good immunogenicity, which could be used as a potential candidate for future HEV vaccine development.

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.

TMEM151A Variants Cause Paroxysmal Kinesigenic Dyskinesia: A Large-Sample Study.

BACKGROUND: Paroxysmal kinesigenic dyskinesia (PKD) is the most common type of paroxysmal dyskinesias. Only one-third of PKD patients are attributed to proline-rich transmembrane protein 2 (PRRT2) mutations. OBJECTIVE: We aimed to explore the potential causative gene for PKD. METHODS: A cohort of 196 PRRT2-negative PKD probands were enrolled for whole-exome sequencing (WES). Gene Ranking, Identification and Prediction Tool, a method of case-control analysis, was applied to identify the candidate genes. Another 325 PRRT2-negative PKD probands were subsequently screened with Sanger sequencing. RESULTS: Transmembrane Protein 151 (TMEM151A) variants were mainly clustered in PKD patients compared with the control groups. 24 heterozygous variants were detected in 25 of 521 probands (frequency = 4.80%), including 18 missense and 6 nonsense mutations. In 29 patients with TMEM151A variants, the ratio of male to female was 2.63:1 and the mean age of onset was 12.93 ± 3.15 years. Compared with PRRT2 mutation carriers, TMEM151A-related PKD were more common in sporadic PKD patients with pure phenotype. There was no significant difference in types of attack and treatment outcome between TMEM151A-positive and PRRT2-positive groups. CONCLUSIONS: We consolidated mutations in TMEM151A causing PKD with the aid of case-control analysis of a large-scale WES data, which broadens the genotypic spectrum of PKD. TMEM151A-related PKD were more common in sporadic cases and tended to present as pure phenotype with a late onset. Extensive functional studies are needed to enhance our understanding of the pathogenesis of TMEM151A-related PKD. © 2021 International Parkinson and Movement Disorder Society.

Case Report: Mutant SCN9A Susceptible to Charcot Neuroarthropathy in a Patient With Congenital Insensitivity to Pain.

Charcot neuroarthropathy is a systemic disease with pathological changes in the musculoskeletal system, which leads to fractures, dislocations, and deformities involving multiple bones and joints, particularly those of the feet. While the common underlying cause of Charcot neuroarthropathy is diabetes mellitus, it is also associated with congenital insensitivity to pain (CIP). CIP is a rare disorder caused by loss-of-function mutations in SCN9A encoding Nav1.7. In this study, we report a patient with CIP from a consanguineous family susceptible to Charcot neuroarthropathy with a novel SCN9A mutation. This report involves the case of a middle-aged man who suffered from CIP, had repeated painless fractures, and developed bone and joint destruction. The physical and radiological examinations revealed that multiple joints were swollen and deformed, and soft-tissue trauma was evident. We identified a novel homozygous SCN9A mutation (p.Cys1339Arg) by whole-exome sequencing (WES), which was verified using Sanger sequencing. In addition, the wild-type (WT) and mutated p. Cys1339Arg were assessed in HEK293 cells expressing Nav1.7, and the results showed that p. Cys1339Arg almost abolished the Nav1.7 sodium current. In conclusion, Charcot neuroarthropathy associated with CIP demonstrated a wider spectrum of Charcot neuroarthropathy than was previously recognized or documented. In addition, this finding is conducive to understanding the critical amino acids for maintaining the function of Nav1.7, thus contributing to the development of Nav1.7-targeted analgesics.

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.

The Phenotypic and Genetic Spectrum of Paroxysmal Kinesigenic Dyskinesia in China.

BACKGROUND: Paroxysmal kinesigenic dyskinesia is a spectrum of involuntary dyskinetic disorders with high clinical and genetic heterogeneity. Mutations in proline-rich transmembrane protein 2 have been identified as the major pathogenic factor. OBJECTIVES: We analyzed 600 paroxysmal kinesigenic dyskinesia patients nationwide who were identified by the China Paroxysmal Dyskinesia Collaborative Group to summarize the clinical phenotypes and genetic features of paroxysmal kinesigenic dyskinesia in China and to provide new thoughts on diagnosis and therapy. METHODS: The China Paroxysmal Dyskinesia Collaborative Group was composed of departments of neurology from 22 hospitals. Clinical manifestations and proline-rich transmembrane protein 2 screening results were recorded using unified paroxysmal kinesigenic dyskinesia registration forms. Genotype-phenotype correlation analyses were conducted in patients with and without proline-rich transmembrane protein 2 mutations. High-knee exercises were applied in partial patients as a new diagnostic test to induce attacks. RESULTS: Kinesigenic triggers, male predilection, dystonic attacks, aura, complicated forms of paroxysmal kinesigenic dyskinesia, clustering in patients with family history, and dramatic responses to antiepileptic treatment were the prominent features in this multicenter study. Clinical analysis showed that proline-rich transmembrane protein 2 mutation carriers were prone to present at a younger age and have longer attack duration, bilateral limb involvement, choreic attacks, a complicated form of paroxysmal kinesigenic dyskinesia, family history, and more forms of dyskinesia. The new high-knee-exercise test efficiently induced attacks and could assist in diagnosis. CONCLUSIONS: We propose recommendations regarding diagnostic criteria for paroxysmal kinesigenic dyskinesia based on this large clinical study of paroxysmal kinesigenic dyskinesia. The findings offered some new insights into the diagnosis and treatment of paroxysmal kinesigenic dyskinesia and might help in building standardized paroxysmal kinesigenic dyskinesia clinical evaluations and therapies. © 2020 International Parkinson and Movement Disorder Society.

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.

A Novel Homozygous SACS Mutation Identified by Whole-Exome Sequencing in a Consanguineous Family with Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a hereditary neurological disorder mostly manifested with a classical triad: progressive early-onset cerebellar ataxia, lower limb pyramidal signs, and peripheral neuropathy. We employed whole-exome sequencing and bioinformatics to identify the genetic cause in an ARSACS patient from a consanguineous family. Based on whole-exome sequences of the patient and her healthy parents, a novel homozygous deletion variant (NM_014363: c.9495_9508del; p.F3166Tfs*9) in the SACS gene was identified in the patient. This frameshift mutation is predicted to generate a truncated sacsin protein, which results in the loss of the C-terminal 1,406 amino acids. Our study provides a potential genetic diagnosis for the patient and expands the spectrum of SACS mutations.

BAG5 Interacts with DJ-1 and Inhibits the Neuroprotective Effects of DJ-1 to Combat Mitochondrial Oxidative Damage.

Loss-of-function mutations in gene encoding DJ-1 contribute to the pathogenesis of autosomal recessive early-onset familial forms of Parkinson's disease (PD). DJ-1 is a multifunctional protein and plays a protective role against oxidative stress-induced mitochondrial damage and cell death, but the exact mechanism underlying this is not yet clearly understood. Here, using coimmunoprecipitation (Co-IP) and immunofluorescence methods, we prove that Bcl-2-associated athanogene 5 (BAG5), a BAG family member, interacts with DJ-1 in mammalian cells. Moreover, we show that BAG5 could decrease stability of DJ-1 and weaken its role in mitochondrial protection probably by influencing dimerization in stress condition. Our study reveals the relationship of BAG5 and DJ-1 suggesting a potential role for BAG5 in the pathogenesis of PD through its functional interactions with DJ-1.

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.

[Metabolic pathways of OGCP and the influence of parkin protein on the metabolism of OGCP].

OBJECTIVE: To study the metabolic pathways of 2-oxoglutarate carrier protein (OGCP)and the influence of parkin protein on the metabolism of OGCP. METHODS: The OGCP metabolic pathways were identified through inhibiting proteasome activities with specific proteasome inhibitors and protease inhibitors. The isotope pulse-chase experiments were performed to measure the turnover rate of OGCP and to study the influence of parkin protein on the metabolism of OGCP. RESULTS: Proteasome inhibitors and protease inhibitors inhibited OGCP degradation. The OGCP metabolism had a half-life of about 8-10 h. Overexpression of parkin protein accelerated the OGCP degradation. CONCLUSION: OGCP degrades through proteasome and lysosome degradation pathways. The degradation of parkin protein can promote the degradation of OGCP.

Genetic variations of Omi/HTRA2 in Chinese patients with Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, with approximately 5-10% of PD cases being linked to genetic factors. The Htra serine peptidase 2 (HTRA2) gene, also known as Omi, was found to be associated with PD in a cohort of German PD patients. However, subsequent studies have indicated that some variants of Omi/HTRA2 may not be related to PD. In order to investigate whether the Omi/HTRA2 gene is related to PD in Han Chinese PD patients, molecular analysis for the Omi/HTRA2 gene was performed in 404 Chinese PD patients and 504 normal individuals. Our present study revealed 2 novel variations. The IVS5+29T>A variant may be a risk factor for PD (P<0.05), while the c.G77A variant might be a pathogenic mutation. However, the findings need to be validated in a larger population using further functional studies.

[Protective effect of over-expression OGCP on HEK293 cells treated by rotenone and mutant Parkin protein].

OBJECTIVE: To study the effect of alpha-ketoglutarate carrier protein (2-oxoglutarate carrier protein, OGCP) and the Parkin protein on HEK293 cell function. METHODS: The cell apoptosis rate, mitochondrial membrane potential and intracellular reactive oxygen species of HEK293 cells treated with rotenone, OGCP and / or Parkin protein were detected by using flow cytometry methods (FCM). RESULTS: (1) Over-expression wild-type Parkin protein and/or OGCP can increase mitochondrial membrane potential of HEK293 cells induced by rotenone, reduce intracellular reactive oxygen species and cell apoptosis rate of HEK293 cells induced by rotenone, while over-expression mutant Parkin (R42P and T240R) protein can decrease the mitochondrial membrane potential of HEK293 cells, especially the HEK293 cells induced by rotenone, but increase intracellular reactive oxygen species and promote apoptosis. (2) In addition, we also found that OGCP can inhibit the increasing of mitochondrial membrane potential and reactive oxygen species and decreasing of cell apoptosis caused by mutant Parkin protein (R42P and T240R). CONCLUSION: (1) Parkin protein and OGCP may be associated with the maintenance of normal function of mitochondria. (2) Over-expression of mutant parkin (R42P and T240R) protein may inhibit mitochondrial function and promote apoptosis. (3) Over-expression OGCP has protective effect on cell toxicity caused by rotenone and mutant parkin protein.

[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.

[Frequency of different subtypes of spinocerebellar ataxia in the Han nationality of Hunan province in China].

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.

[Clinical characteristics and molecular biology of hereditary spinocerebellar ataxia type 7: study of 3 Chinese families].

OBJECTIVE: To study the clinical characteristics and molecular biology of hereditary spinocerebellar ataxia type 7 (SCA7). METHODS: Peripheral blood samples were collected from 245 with autosomal dominant SCA from 184 families and 71 sporadic SCA patients. Polymerase chain reaction, polyacrylamide gel electrophoresis, and capillary electrophoresis technique were used to detect the SCA7 (CAG) n trinucleotide repeat mutations. 163 healthy persons were used as controls. The abnormal allele fragments were sequenced by ABI 377 DNA sequencing machine. RESULTS: Three SCA families with 15 patients were identified with a positive rate of 1.6%. DNA sequencing showed that the abnormal SCA7 alleles with CAG repeat were expanded to 38 to 71 repeats, and the normal SCA7 alleles were carried from 6 to 15 CAG repeats. Analysis of parent-child couples demonstrated the existence of marked anticipation in 2 families, especially in paternal transmission. Linkage analysis found a maximum two-point LOD score of 2.82 in the microsatellite D3S1300 at recombination fraction (theta = 0.00). CONCLUSION: CAG expansion is the pathogenic cause of SCA7, a rare subtype of SCA. The 38 CAG is the minimum pathological expansion in mainland China.

[Screening for proteins interacting with ataxin-3, the gene product of SCA3/MJD].

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.

[The wild-type alpha-synuclein over-expression to induce the protein aberrant aggregation of alpha-synuclein in HEK293 cells in vitro].

OBJECTIVE: To investigate over-expression of wild-type alpha-synuclein inducing the aberrant aggregation of alpha-synuclein in HEK293 cell in vitro. METHODS: The cDNA encoding the human alpha-synuclein without the stop code was cloned into PGEM T-easy vector. Using enzyme map and DNA sequencing analyzed and determined the recombinant plasmid, and then sub-clone the alpha-synuclein cDNA fragment into pEGFP-N1 vector. The recombinant plasmids alpha-synuclein-pEGFP were transfected into HEK293 cells by lipofectamin 2000. The aberrant aggregation of alpha-synuclein was measured by EGFP fluorescence, anti-alpha-synuclein immunocytochemistry. The inclusions in the cultured cells were identified with HE staining. RESULTS: The restriction enzyme map suggested that eukaryotic expression vector for human wild-type alpha-synuclein gene was constructed successfully. By EGFP fluorescence, anti-alpha-synuclein immunocytochemistry, it could be observed that the alpha-synuclein protein could aggregate in cytoplasm and the Lewy body-like inclusions found in cytoplasm of cultured cells. CONCLUSION: The over-expression of wild-type alpha-synuclein can induce protein aberrant aggregation and Lewy body-like inclusions formation in cytoplasm of HEK293 cell in vitro.

[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.