Neuronal small extracellular vesicles carrying miR-181c-5p contribute to the pathogenesis of epilepsy by regulating the protein kinase C-δ/glutamate transporter-1 axis in astrocytes.

Information exchange between neurons and astrocytes mediated by extracellular vesicles (EVs) is known to play a key role in the pathogenesis of central nervous system diseases. A key driver of epilepsy is the dysregulation of intersynaptic excitatory neurotransmitters mediated by astrocytes. Thus, we investigated the potential association between neuronal EV microRNAs (miRNAs) and astrocyte glutamate uptake ability in epilepsy. Here, we showed that astrocytes were able to engulf epileptogenic neuronal EVs, inducing a significant increase in the glutamate concentration in the extracellular fluid of astrocytes, which was linked to a decrease in glutamate transporter-1 (GLT-1) protein expression. Using sequencing and gene ontology (GO) functional analysis, miR-181c-5p was found to be the most significantly upregulated miRNA in epileptogenic neuronal EVs and was linked to glutamate metabolism. Moreover, we found that neuronal EV-derived miR-181c-5p interacted with protein kinase C-delta (PKCδ), downregulated PKCδ and GLT-1 protein expression and increased glutamate concentrations in astrocytes both in vitro and in vivo. Our findings demonstrated that epileptogenic neuronal EVs carrying miR-181c-5p decrease the glutamate uptake ability of astrocytes, thus promoting susceptibility to epilepsy.

Encephalitis-like episodes with cortical edema and enhancement in patients with neuronal intranuclear inclusion disease.

OBJECTIVES: Neuronal intranuclear inclusion disease (NIID) exhibited significant clinical heterogeneities. However, the clinical features, radiographic changes, and prognosis of patients with encephalitis-like NIID have yet to be systematically elucidated. METHODS: Clinical data including medical history, physical examination, and laboratory examinations were collected and analyzed. Skin and sural nerve biopsies were conducted on the patient. Repeat-primed PCR (RP-PCR) and fluorescence amplicon length PCR (AL-PCR) were used to detect the expansion of CGG repeat. We also reviewed the clinical and genetic data of NIID patients with cortical enhancement. RESULTS: A 54-year-old woman presented with encephalitis-like NIID, characterized by severe headache and agitative psychiatric symptoms. The brain MRI showed cortical swelling in the temporo-occipital lobes and significant enhancement of the cortical surface and dura, but without hyperintensities along the corticomedullary junction on diffusion-weighted image (DWI). A biopsy of the sural nerve revealed a demyelinating pathological change. The intranuclear inclusions were detected in nerve and skin tissues using the p62 antibody and electron microscopy. RP-PCR and AL-PCR unveiled the pathogenic expansion of CGG repeats in the NOTCH2NLC gene. A review of the literature indicated that nine out of the 16 patients with cortical lesions and linear enhancement exhibited encephalitis-like NIID. CONCLUSION: This study indicated that patients with encephalitis-like NIID typically exhibited headache and excitatory psychiatric symptoms, often accompanied by cortical edema and enhancement of posterior lobes, and responded well to glucocorticoid treatment. Furthermore, some patients may not exhibit hyperintensities along the corticomedullary junction on DWI, potentially leading to misdiagnosis.

Whole exome sequencing indicating GGCCTG hexanucleotide repeat in patients with spinocerebellar ataxia type 36.

INTRODUCTION: Spinocerebellar ataxia type 36 (SCA36) is caused by large GGCCTG repeat expansion in the NOP56 gene. The genetic diagnosis based on Southern blot is expensive and time-consuming. This study aimed to evaluate the reliability and effectiveness of whole exome sequencing (WES) for routine genetic diagnosis of suspected SCA36 patients. METHODS: Pathogenic repeat expansions for SCAs including SCA36 were first analyzed based on WES data using ExpansionHunter in five probands from SCA families, then the results were confirmed by triplet repeat primed polymerase chain reaction (TP-PCR) and Southern blot. RESULTS: GGCCTG repeat expansion in NOP56 was indicated in all five probands by WES, then it was found in 11 SCA patients and three asymptomatic individuals by TP-PCR. The sizes of GGCCTG repeat expansions were confirmed to be 1390-1556 by Southern blot. The mean age at onset of the patients was 51.0 ± 9.3 (ranging from 41 to 71), and they presented slowly progressive cerebellar ataxia, atrophy and fasciculation in tongue or limb muscles. CONCLUSION: The patients were clinically and genetically diagnosed as SCA36. This study proposed that WES could be a rapid, reliable, and cost-effective routine test for the preliminarily detection of SCA36 and other ataxia diseases.

CAG Repeat Expansion in THAP11 Is Associated with a Novel Spinocerebellar Ataxia.

BACKGROUND: More than 50 loci are associated with spinocerebellar ataxia (SCA), and the most frequent subtypes share nucleotide repeats expansion, especially CAG expansion. OBJECTIVE: The objective of this study was to confirm a novel SCA subtype caused by CAG expansion. METHODS: We performed long-read whole-genome sequencing combined with linkage analysis in a five-generation Chinese family, and the finding was validated in another pedigree. The three-dimensional structure and function of THAP11 mutant protein were predicted. Polyglutamine (polyQ) toxicity of THAP11 gene with CAG expansion was assessed in skin fibroblasts of patients, human embryonic kidney 293 and Neuro-2a cells. RESULTS: We identified THAP11 as the novel causative SCA gene with CAG repeats ranging from 45 to 100 in patients with ataxia and from 20 to 38 in healthy control subjects. Among the patients, the number of CAA interruptions within CAG repeats was decreased to 3 (up to 5-6 in controls), whereas the number of 3' pure CAG repeats was up to 32 to 87 (4-16 in controls), suggesting that the toxicity of polyQ protein was length dependent on the pure CAG repeats. Intracellular aggregates were observed in cultured skin fibroblasts from patients. THAP11 polyQ protein was more intensely distributed in the cytoplasm of cultured skin fibroblasts from patients, which was replicated with in vitro cultured neuro-2a transfected with 54 or 100 CAG repeats. CONCLUSIONS: This study identified a novel SCA subtype caused by intragenic CAG repeat expansion in THAP11 with intracellular aggregation of THAP11 polyQ protein. Our findings extended the spectrum of polyQ diseases and offered a new perspective in understanding polyQ-mediated toxic aggregation. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

A new phenotype of syndromic retinitis pigmentosa with myopathy is caused by mutations in retinol dehydrogenase 11.

Retinol dehydrogenase 11 (RDH11) is an 11-cis-retinol dehydrogenase that has a well-characterized, albeit auxiliary role in the retinoid cycle. Diseases caused by mutations in the RDH11 gene are very rare, and only one affected family with eye and intelligence involvement has been reported. In the present study, we describe the clinical and genetic findings in a Chinese patient with retinitis pigmentosa (RP), juvenile cataracts, intellectual disability, and myopathy. Trio-based whole-exome sequencing and whole genomic copy number variation detection were performed in this family, and compound heterozygous mutations were identified in RDH11 of the patient: c.938T>C (p.Leu313Pro) derived from the father and c.75-3C>A derived from the mother. Variant c.75-3C>A was confirmed to be a splice-site mutation by cDNA sequencing. It caused exon 2 skipping, resulting in a frameshift mutation and premature translation termination (p.Lys26Serfs*38). Moreover, we found mislocalization of RDH11 protein in muscle cells of the patient by using immunofluorescence staining. This is the first case reported in the Chinese population harboring mutations in RDH11 and revealing a new phenotype of syndromic RP with myopathy.

Clinical spectrum and genetic variations of LMNA-related muscular dystrophies in a large cohort of Chinese patients.

BACKGROUND: LMNA-related muscular dystrophy is caused by mutations in LMNA gene. We aimed to identify genetic variations and clinical features in a large cohort of Chinese patients with LMNA mutations in an attempt to establish genotype-phenotype correlation. METHODS: The clinical presentations of patients with LMNA-related muscular dystrophy were recorded using retrospective and prospective cohort study. LMNA mutation analysis was performed by Sanger sequencing or next-generation sequencing. Mosaicism was detected by personal genome machine amplicon deep sequencing for mosaicism. RESULTS: Eighty-four patients were identified to harbour LMNA mutations. Forty-one of those were diagnosed with LMNA-related congenital muscular dystrophy (L-CMD), 32 with Emery-Dreifuss muscular dystrophy (EDMD) and 11 with limb-girdle muscular dystrophy type 1B (LGMD1B). We identified 21 novel and 29 known LMNA mutations. Two frequent mutations were identified: c.745C>T and c.1357C>T. A correlation between the location of mutation and the clinical phenotype was observed: mutations affecting the head and coil 2A domains mainly occurred in L-CMD, while the coil 2B and Ig-like domains mainly related to EDMD and LGMD1B. We found somatic mosaicism in one parent of four probands. Muscle biopsies revealed 11 of 20 biopsied L-CMD exhibited inflammatory changes, and muscle cell ultrastructure showed abnormal nuclear morphology. CONCLUSIONS: Our detailed clinical and genetic analysis of 84 patients with LMNA-related muscular dystrophy expands clinical spectrum and broadens genetic variations caused by LMNA mutations. We identified 21 novel and 29 known LMNA mutations and found two frequent mutations. A correlation between the location of mutation and the clinical severity was observed. Preliminary data suggested that low-dose corticosteroid treatment may be effective.

Establishment of a comprehensive method to derive seawater quality criteria of BDE-47 in China.

2,2',4,4'-tetrabromodiphenyl ether (BDE-47) is an emerging toxic organic pollutant widely detected in territorial waters. Accordingly, establishing the seawater quality criteria (SWQC) for BDE-47 is of significant importance to protect the marine ecosystems. In this study, published ecotoxicity data of BDE-47 to aquatic species in China were collected, and acute and chronic toxicity tests were carried out on saltwater aquatic organisms from 5 phyla and 8 families widely existed in the Chinese marine environment. Static acute toxicity tests and renewed chronic toxicity tests were adopted. The scientific theories and technical methods of the United States Environmental Protection Agency (US EPA), Canada, the European Union (EU), and the Netherlands on water quality criteria guidelines, as well as the Chinese freshwater quality criteria guidelines were compared. Then an integrated method of SWQC derivation was introduced through comprehensive consideration. Afterward, the SWQC of BDE-47 was derived based on the ecotoxicity data. The SWQC includes short-term seawater quality criteria (S-SWQC), long-term seawater quality criteria (L-SWQC), and serious risk concentration for the ecosystem (SRCeco). And they were derived and recommended as 7.90 µg/L, 0.217 µg/L, and 3.65 µg/L, respectively. This study served as a specific example to quantitatively studies the differences between different scientific theories and technique methods. The derivation process and improvement of SWQC for BDE-47 provide support for the future revision of water quality criteria in China.

The expression levels of Mir-146b and Mir-221 in thyroid carcinoma tissues and their correlation with malignancy degree.

This experiment aimed to investigate the correlation between the expressions of mir-146b and mir-221 and the proliferation, invasion, and malignancy of tumor cells. For this purpose, 135 patients with thyroid cancer treated in our hospital and 120 patients with non-malignant thyroid cancer in the outpatient clinic were selected as subjects. Fine needle biopsy tissues of the two groups were taken as samples to detect the expression levels of mir-146b and mir-221 in the tissues. The effect of the two microRNA on the proliferation and migration of cancer cells was observed by the transfection of cell lines. The contents of gal-3 and MMP9 in serum were further detected, and their relationship with the expressions of mir-146b and mir-221 was analyzed. The results showed that the expressions of mir-146b and mir-221 were significantly decreased and increased in thyroid carcinoma fine-needle aspiration tissues. Mir-146b inhibited the proliferation and migration of cancer cells, while mir-221 promoted this process. In the more cancer cells, the expression levels of these two genes changed more, and the serum levels of gal-3 and MMP9 also increased. It was concluded that the expression levels of mir-146b and mir-221 were correlated with the degree of tumor malignancy, and the low expression of mir-146b and the high expression of mir-221 were correlated with the proliferation, invasion, and malignancy of thyroid cancer tumor cells.

Nuclear Factor-κB Pathway Mediates the Molecular Pathogenesis of LMNA-Related Muscular Dystrophies.

LMNA-related muscular dystrophies are caused by mutations of the LMNA gene. Inflammatory changes and cellular apoptosis are significant pathological findings in the muscle cells of these patients. We aimed to investigate the roles of nuclear factor-κB (NF-κB) mediated inflammation as a molecular mechanism for the pathogenesis of LMNA-related muscular dystrophies. Muscle specimen of a patient with LMNA gene mutation (c.1117A>G, p.I373V, reported in our previous work) showed significant inflammatory changes. The ultrastructure of muscle cells showed severe nuclear abnormalities compared with the control. Therefore, we used this mutation to establish mutant cell line for in vitro studies. Transfected human embryonic kidney 293 (HEK293) cells containing a mutant construct from this patient showed irregular nuclear morphology. Mass spectrometry analysis suggested genomic instability and augmented expression of apoptosis-related genes. We detected activation of NF-κB pathway in LMNA mutant cells which promoted the expression of downstream inflammatory factors. The LMNA mutation also activated the molecular pathway of apoptosis in LMNA mutant cells. These are important molecular mechanisms underlying the pathogenesis of LMNA-related muscular dystrophies. Our research provides crucial evidence for future pathogenetic studies and possible treatment strategies for LMNA-related muscular dystrophies.

Peripheral Nerve Injury-Induced Astrocyte Activation in Spinal Ventral Horn Contributes to Nerve Regeneration.

Accumulating evidences suggest that peripheral nerve injury (PNI) may initiate astrocytic responses in the central nervous system (CNS). However, the response of astrocytes in the spinal ventral horn and its potential role in nerve regeneration after PNI remain unclear. Herein, we firstly illustrated that astrocytes in the spinal ventral horn were dramatically activated in the early stage following sciatic nerve injury, and these profiles were eliminated in the chronic stage. Additionally, we found that the expression of neurotrophins, including brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin-3 (NT-3), also accompanied with astrocyte activation. In comparison with the irreversible transected subjects, astrocyte activation and the neurotrophic upregulation in the early stage were more drastic in case the transected nerve was rebridged immediately after injury. Furthermore, administering fluorocitrate to inhibit astrocyte activation resulted in decreased neurotrophin expression in the spinal ventral horn and delayed axonal regeneration in the nerve as well as motor function recovery. Overall, the present study indicates that peripheral nerve injury can initiate astrocyte activation accompanied with neurotrophin upregulation in the spinal ventral horn. The above responses mainly occur in the early stage of PNI and may contribute to nerve regeneration and motor function recovery.

[Clinical and genetic study of a Chinese family affected with caveolinopathies].

OBJECTIVE: To analyze clinical features and genetic mutations in a Chinese family affected with autosomal dominant caveolinopathies. METHODS: Clinical data of the proband and her family members were collected. Genomic DNA was extracted from peripheral blood samples with a standard procedure. Next generation sequencing was carried out for the proband, and direct sequencing was employed to detect potential mutation of the CAV gene. RESULTS: The proband presented with slowly progressing distal muscle weakness and atrophy, especially distal upper limbs and muscular soreness during early childhood, with her CK level moderately elevated and EMG showing myogenic and neurogenic injuries. Her sisters presented mild symptoms with hand muscle atrophy and fasciculation after exercise. A heterozygous missense mutation c.80G>A (p.Arg27Gln), which was reported as being pathogenic, was identified in the CAV3 gene in the proband and her sisters. CONCLUSION: A heterozygous c.80G>A (p.Arg27Gln) mutation in the CAV3 gene probably underlies the autosomal dominant caveolinopathies in this Chinese family.

Homozygous variant in COQ7 causes autosomal recessive hereditary spastic paraplegia.

Biallelic mutations in the coenzyme Q7 (COQ7) encoding gene were recently identified as a genetic cause of distal hereditary motor neuropathy. Here, we explored the clinical, electrophysiological, pathological, and genetic characteristics of a Chinese patient with spastic paraplegia associated with recessive variants in COQ7. This patient carried a novel c.322C>A (p.Pro108Thr) homozygous variant. Sural biopsy revealed mild mixed axonal and demyelinating degeneration. Immunoblotting showed a significant decrease in the COQ7 protein level in the patient's fibroblasts. This study confirmed that COQ7 variant as a genetic cause of HSP, and further extended spastic paraplegia to the phenotypic spectrum of COQ7-related disorders.

Sorbs2 regulates seizure activity by influencing AMPAR-mediated excitatory synaptic transmission in temporal lobe epilepsy.

Temporal lobe epilepsy (TLE), the most common type of drug-resistant epilepsy, severely affects quality of life. However, the underlying mechanism of TLE remains unclear and deserves further exploration. Sorbs2, a key synaptic regulatory protein, plays an important role in the regulation of synaptic transmission in the mammalian brain. In this study, we aimed to investigate the expression pattern of Sorbs2 in a kainic acid (KA)-induced TLE mouse model and in patients with TLE to further determine whether Sorbs2 is involved in seizure activity and to explore the potential mechanism by which Sorbs2 affects seizures in this TLE mouse model. First, we found that the expression of Sorbs2 was obviously increased in the hippocampus and cortex of a TLE mouse model and in the temporal cortex of TLE patients, indicating an abnormal expression pattern of Sorbs2 in TLE. Importantly, subsequent behavioral analyses and local field potential (LFP) analyses of a TLE mouse model demonstrated that the downregulation of hippocampal Sorbs2 could prolong the latency to spontaneous recurrent seizures (SRSs) and protect against SRSs. We also found that the knockdown of Sorbs2 in the hippocampus could decrease excitatory synaptic transmission in pyramidal neurons (PNs) in the hippocampal CA1 region and reduce the expression levels of the AMPAR subunits GluA1 and GluA2. Thus, we speculated that Sorbs2 may promote epileptogenesis and the development of TLE by affecting AMPAR-mediated excitatory synaptic transmission in PNs in the CA1 region. Therefore, reducing the expression of hippocampal Sorbs2 could restrain epileptogenesis and the development of TLE.

Clinical and genetic characteristics of myotonia congenita in Chinese population.

Myotonia congenita (MC) is a rare hereditary muscle disease caused by variants in the CLCN1 gene. Currently, the correlation of phenotype-genotype is still uncertain between dominant-type Thomsen (TMC) and recessive-type Becker (BMC). The clinical data and auxiliary examinations of MC patients in our clinic were retrospectively collected. Electromyography was performed in 11 patients and available family members. Whole exome sequencing was conducted in all patients. The clinical and laboratory data of Chinese MC patients reported from June 2004 to December 2022 were reviewed. A total of 11 MC patients were included in the study, with a mean onset age of 12.64 ± 2.73 years. The main symptom was muscle stiffness of limbs. Warm-up phenomenon and percussion myotonia were found in all patients. Electromyogram revealed significant myotonic charges in all patients and two asymptomatic carriers, while muscle MRI and biopsy showed normal or nonspecific changes. Fourteen genetic variants including 6 novel variants were found in CLCN1. Ninety-eight Chinese patients were re-analyzed and re-summarized in this study. There were no significant differences in the demographic data, clinical characteristics, and laboratory findings between 52 TMC and 46 BMC patients. Among the 145 variants in CLCN1, some variants, including the most common variant c.892 G>A, could cause TMC in some families and BMC in others. This study expanded the clinical and genetic spectrum of Chinese patients with MC. It was difficult to distinguish between TMC and BMC only based on the clinical, laboratory, and genetic characteristics.

Lama1 upregulation prolongs the lifespan of the dyH/dyH mouse model of LAMA2-related congenital muscular dystrophy.

LAMA2-related congenital muscular dystrophy (LAMA2-CMD), characterized by laminin-α2 deficiency, is debilitating and ultimately fatal. To date, no effective therapy has been clinically available. Laminin-α1, which shares significant similarities with laminin-α2, has been proven as a viable compensatory modifier. To evaluate its clinical applicability, we establish a Lama2 exon-3 deletion mouse model (dyH/dyH). The dyH/dyH mice exhibit early lethality and typical LAMA2-CMD phenotypes, allowing the evaluation of various endpoints. In dyH/dyH mice treated with synergistic activation mediator-based CRISPRa-mediated Lama1 upregulation, a nearly doubled median survival is observed, as well as improvements in weight and grip. Significant therapeutical effects are revealed by MRI, serum biochemical indices, and muscle pathology studies. Treating LAMA2-CMD with LAMA1 upregulation is feasible and that early intervention can alleviate symptoms and extend lifespan. Additionally, we reveal limitations of LAMA1 upregulation, including high-dose mortality and non-sustained expression, which require further optimization in future studies.

Skin biopsy and neuronal intranuclear inclusion disease.

Neuronal intranuclear inclusion disease (NIID) is a rare neurodegenerative disease with variable clinical phenotypes. There is a considerable delay in the definite diagnosis, which primarily depends on postmortem brain pathological examination. Although CGG repeat expansion in the 5'-untranslated region of NOTCH2NLC has been identified as a disease-associated variant, the pathological diagnosis is still required in certain NIID cases. Intranuclear inclusions found in the skin tissue of patients with NIID dramatically increased its early detection rate. Skin biopsy, as a minimally invasive method, has become widely accepted as a routine examination to confirm the pathogenicity of the repeat expansion in patients with suspected NIID. In addition, the shared developmental origin of the skin and nerve system provided a new insight into the pathological changes observed in patients with NIID. In this review, we systematically discuss the role of skin biopsy for NIID diagnosis, the procedure of skin biopsy, and the pathophysiological mechanism of intranuclear inclusion in the skin.

Uniparental disomy for chromosome 1 with POMGNT1 splice-site variant causes muscle-eye-brain disease.

POMGNT1, encoding protein O-mannose beta-1,2-N-acetylglucosaminyltransferase 1, is one of the genes responsible for dystroglycanopathy (DGP), which includes multiple phenotypes such as muscle-eye-brain disease (MEB), congenital muscular dystrophy with intellectual disability, and limb-girdle muscular dystrophy Here, we report a case of MEB that is the result of a homozygous variant of POMGNT1 that is revealed through uniparental disomy (UPD). An 8-month-old boy was admitted with mental and motor retardation, hypotonia, esotropia, early onset severe myopia, and structural brain abnormalities. A panel testing of genetic myopathy-related genes was used to identify a homozygous c.636C>T (p.Phe212Phe) variant in exon 7 of POMGNT1 in the patient, a heterozygous c.636C>T variant in the father, and the wild type in the mother. Quantitative polymerase chain reaction (q-PCR) revealed no abnormal copy numbers in exon 7. Trio-based whole-exome sequencing (trio-WES) revealed a possible paternal UPD on chromosome 1 of the patient. Chromosomal microarray analysis (CMA) revealed a 120,451 kb loss of heterozygosity (LOH) on 1p36.33-p11.2, encompassing POMGNT1, and a 99,319 kb loss of heterozygosity on 1q21.2-q44, which indicated UPD. Moreover, RNA sequencing (RNA-seq) verified that the c.636C>T variant was a splice-site variant, leading to skipping of exon 7 (p.Asp179Valfs*23). In conclusion, to the best of our knowledge, we present the first case of MEB caused by UPD, providing valuable insights into the genetic mechanisms underlying this condition.

Unique genotype-phenotype correlations within LAMA2-related limb girdle muscular dystrophy in Chinese patients.

Background: LAMA2-related limb girdle muscular dystrophy (LGMD R23) is rare. The detailed clinical phenotypes and genetic information associated with LGMD R23 are unknown. Methods: We conducted a retrospective cross-sectional and longitudinal study on 19 LGMD R23 patients. Results: Normal early motor development was observed in 84.2% patients. Mild orthopedic complications were observed in 42.1% patients. 36.8% patients had seizures, which is unusually frequent in LGMD. Epilepsy was eventually diagnosed in 26.3% patients. 46.7% patients presented with motor neuropathy. Genetic analysis identified 29 pathogenic variants, with missense and frameshift variants being the most common. The mutant sites were mainly distributed in the N-terminal and G-like domains of laminin. The missense variants are distributed near the N-terminus (exons 3-11), whereas frameshift variants are distributed in exons 12-65. Five patients were diagnosed with epilepsy and all of them harbor at least one missense variants in exon 4. 71.4% variants of patients with motor neuropathy located in the LN domain. Conclusions: Missense variants in exon 4 maybe correlated with epilepsy and variants in the LN domain maybe correlated with motor neuropathy in Chinese patients. Our study expands the clinical and genetic spectrum caused by LAMA2 variations and provides novel genotype-phenotype correlations of LGMD R23.