Phenotypic and genotypic analysis of a patient with Miyoshi myopathy caused by truncated protein.

Dysferlin protein deficiency can cause neuromuscular dysfunction, resulting in autosomal recessive dysferlinopathy, which is caused by DYSF gene mutation. Dysferlin proteins belongs to the Ferlin1-like protein family and are associated with muscle membrane repair and regeneration. In China, pathogenic mutations of the protein often result in two clinical phenotypes of Miyoshi muscular or limb band muscular dystrophy type 2B. It is clinically characterized by progressive muscle weakness and elevated serum creatine kinase. The data of the child were collected, blood samples of the child and his family members were collected, and whole exome sequencing (WES) was performed. The recombinant expression vector was constructed, the function of the mutation was verified by minigene, and the pathogenicity of the mutation was further analyzed by combining with biological information analysis. The patient initially presented with asymptomatic elevation of serum creatine kinase（CK）. Then progressive lower limb weakness, mainly distal limb weakness. Large amounts of scattered necrosis, myogenic lesions, and complete deletion of dysferlin protein were observed under muscle biopsy, which further improved genetic detection. Whole exome sequencing showed compound mutations (c.1397 + 1_1397 + 3del and c.1375dup p.M459Nfs*15) in DYSF gene. c.1375dup p.M459Nfs*15 have been reported. The other mutation is the deletion of c.1397 + 1_1397 + 3 in Intron15, which is an intron mutation that may affect splicing and the pathogenesis is still unknown. Minigene splicing assay verified that c.1397 + 1_1397 + 3del resulted in exon15 skipping and produced a premature termination codon. We report a novel pathogenic mutation in DYSF gene with Miyoshi myopathy and demonstrate this variant causes skipping of exon15 by minigene splicing assay. We point out the need of conducting functional analysis to verify the pathogenicity of intronic mutation. The finding enriches the mutation spectrum of DYSF gene and laid a foundation for future studies on the correlation between genotype and phenotype.

SCAF4 variants are associated with epilepsy with neurodevelopmental disorders.

AIMS: The genetic causes of epilepsy with unknown etiology in most patients remain unknown. The aim of this study was to elucidate the phenotype of SCAF4-related epilepsy. METHODS: Trio-based whole-exome sequencing was performed in patients with epilepsy. Silico programs and protein modeling were employed to predict the damaging of variants. Previously reported SCAF4 variants were systematically reviewed to analyze the genotype-phenotype correlations. RESULTS: Three heterozygous variants in the SCAF4 were detected in three cases, including one missense variant and two frameshift variants. All variants were de novo. None of these variants is present in gnomAD controls. The missense variant was predicted to be damaging in silico tools. Protein modeling showed that two frameshift variants resulted in loss of domains, and the missense variant may disrupt a nearby phosphorylation site and alter the hydrogen bonds around 54C and the stability of the SCAF4 protein. Intellectual development was mildly delayed for all patients except for one with whom contact was lost. All probands experienced epilepsy as infrequent seizures, responded well to antiseizure drugs, and had a median [IQR] seizure onset age of 4 [1.75, 7.5] years. The variants in the domain-encoding exons and upstream exons exhibited a strong association with epilepsy. CONCLUSIONS: SCAF4 is a potential causative gene of epilepsy with neurodevelopmental disorders.

Perampanel and lacosamide monotherapy in pediatric patients with newly diagnosed focal epilepsy: A prospective study evaluating efficacy, tolerability, and behavior.

PURPOSE: Perampanel (PER) and lacosamide (LCM) are the new third-generation anti-seizure medications (ASMs) that were approved for the monotherapy of focal epilepsy in children over four years of age in China, in 2021. Very few studies have analyzed the application of PER monotherapy among pediatric patients aged ≥four years, and no study compared the efficacy and tolerability of PER monotherapy with LCM monotherapy in pediatric patients with focal epilepsy. The present study aimed to investigate the efficacy, tolerability, and effect on behavior and emotion of PER and LCM as monotherapy in pediatric patients with newly diagnosed focal epilepsy, which is beneficial for clinicians to have more choices to treat pediatric patients with focal epilepsy. METHODS: This was a prospective, single-center, observational study that involved pediatric patients (disease onset age ≥four years) with newly diagnosed focal epilepsy treated with PER or LCM as primary monotherapy. Outcomes included retention, being responders, and seizure-free rates after 3, 6, and 12 months. Adverse events (AEs) were noticed throughout the follow-up period. Behavioral outcomes were evaluated with Achenbach Child Behavior Checklist (CBCL/4-16) at baseline and after three and six months. RESULTS: Using randomization, 60 patients receiving PER (31 females, 29 males, median age: 7.79 [5.34, 10.16] years, median dose: 3.0 [2.0, 4.0] mg/day) and 60 patients receiving LCM (25 females, 35 males, median age: 7.72 [5.91, 10.72] years, median dose: 150.0 [100.0, 200.0] mg/day) were enrolled in the study. At the 12-month follow-up, the retention rates in the PER and LCM groups, both were 90.4%, and the responder rates were 65.4% and 71.2%, while seizure-free rates were 57.7% and 67.3%, respectively. There were no significant differences in the retention, responder and seizure-free rates between the two groups (P > 0.05). There were no significant differences in the responder rates between patients with BECTS, abnormal brain magnetic resonance imaging (MRI), or types of seizure in the two groups (P > 0.05). In the PER group, 28.8% (15/52) of patients experienced AEs, of which the most frequently reported were irritability (n = 7; 13.5%), dizziness (n = 5; 9.6%), somnolence (n = 3; 5.8%), ataxia (n = 1; 1.9%), headache (n = 1; 1.9%), and rash (n = 1; 1.9%). In the LCM group, 15.4% (8/52) of the patients had AEs, including headache (n = 4; 7.5%), dizziness (n = 4; 7.5%), nausea (n = 2; 3.8%), somnolence (n = 2; 3.8%), irritability (n = 1; 1.9%), stomach ache (n = 1; 1.9%), and vomiting (n = 1; 1.9%). The incidence of irritability was significantly higher in the PER group than in the LCM group (13.5% vs. 1.9%, P = 0.031), which occurred mainly within eight weeks after drug administration. Patients with irritability were not dangerous to surrounding people by the assessment of parental observation in the life. And the symptoms were relieved spontaneously within a few months. The outcomes of total scores, internalizing scores, and externalizing scores of the CBCL did not show statistically significant differences in the PER and LCM groups between baseline and three and six months. Characteristics of behavior and emotion did not have substantial changes in patients treated with PER and LCM monotherapy. CONCLUSIONS: The present study documented similar good effectiveness and good tolerance of PER and LCM as monotherapy in pediatric patients with newly diagnosed focal epilepsy and showed no behavioral or emotional impact, as assessed by the CBCL. Though the incidence of irritability with PER monotherapy may be higher than that with LCM monotherapy soon after medication initiation, this side effect appears to resolve spontaneously within a few months. At present, this study was the first research about PER and LCM monotherapy in pediatric patients with newly diagnosed focal epilepsy evaluating efficacy, tolerability, and behavior in China.

Therapeutic Drug Monitoring of Perampanel in Children With Refractory Epilepsy: Focus on Influencing Factors on the Free-Perampanel Concentration.

BACKGROUND: This study aimed to assess the effect of perampanel dose, age, sex, and antiseizure medication cotherapy on steady-state free-perampanel concentration in children with refractory epilepsy, as well as the relationship between inflammation and the pharmacokinetics of perampanel. METHODS: This prospective study in China included 87 children with refractory epilepsy treated with adjunctive perampanel therapy. Free and total perampanel concentrations in plasma were determined using liquid chromatography-tandem mass spectrometry. Free-perampanel concentration was compared among patients with various potential influencing factors. RESULTS: A total of 87 pediatric patients (44 female children) aged 2-14 years were enrolled. The mean free-perampanel concentration and free concentration-to-dose (CD) ratio in plasma were 5.7 ± 2.7 ng/mL (16.3 ± 7.7 nmol/L) and 45.3 ± 21.0 (ng/mL)/(mg/kg) [129.6 ± 60.1 (nmol/L)/(mg/kg)], respectively. The protein binding of perampanel in plasma was 97.98%. A linear relationship was observed between perampanel dose and free concentration in plasma, and a positive relationship was found between the total and free-perampanel concentrations. Concomitant use of oxcarbazepine reduced the free CD ratio by 37%. Concomitant use of valproic acid increased the free CD ratio by 52%. Five patients had a plasma high-sensitivity C-reactive protein (Hs-CRP) level of >5.0 mg/L (Hs-CRP positive). The total and free CD ratios of perampanel were increased in patients with inflammation. Two patients with inflammation developed adverse events, which disappeared as the Hs-CRP level returned to normal, and neither required perampanel dose reduction. Age and sex did not influence the free-perampanel concentration. CONCLUSIONS: This study found complex drug interactions between perampanel and other concomitant antiseizure medications, providing valuable information to enable clinicians to apply perampanel in the future reasonably. In addition, it may be important to quantify both the total and free concentrations of perampanel to assess complex pharmacokinetic interactions.

Treating GNAO1 mutation-related severe movement disorders with oxcarbazepine: a case report.

Background: GNAO1 variants have been found to be associated with epileptic encephalopathies, developmental delays (DDs), and movement disorders (MDs). Therapies for patients with GNAO1 variants vary. However, treatments for GNAO1-related diseases are still in their infancy. Previous reports suggest that few pharmacological treatments are effective for patients with GNAO1 variant-related MDs. Deep brain stimulation (DBS) treatment appears to be effective, however surgical procedures and equipment failures pose risks to the patients. Effectiveness for oxcarbazepine (OXC) in GNAO1 variant-related MDs is first reported in our study, and it expand the effective drugs for MD treatment. Case Description: We report the case of a 5-year-old male patient with a MD, who suffered from hypotonia and refractory choreoathetosis. The patient was found to have a DD and an intellectual disability. A de-novo variant of the GNAO1 gene (NM_138736: exom6: c.709G>A [p. Glu237Lys]) was identified by whole exome sequencing (WES) when he was 8 months old. The patient visited our hospital at the age of 4 years and 3 months because of fever and recurrent convulsions. Electroencephalogram (EEG) results show abnormal spikes, and magnetic resonance imaging (MRI) showed the enlargement of the lateral ventricles. The administration of tiapride hydrochloride, phenobarbital, midazolam, and hormones had no effect. OXC treatment was then initiated. No MD behaviors, such as rigidity and twisting of the limbs and trunk, or chorea, were observed after 10 days OXC treatment. Eventually, incremental doses of OXC were effective, and our patient achieved good control of his MD. Conclusions: We are the first to demonstrate the role of OXC in alleviating MDs associated with GNAO1 mutations. This report provides a novel possibility for the clinical treatment of this rare disease. To manage MDs associated with GNAO1 mutations, we recommend that OXC treatment be attempted before invasive surgical therapy.

Development and validation of a nomogram for the early prediction of drug resistance in children with epilepsy.

Background and purpose: This study aimed to effectively identify children with drug-resistant epilepsy (DRE) in the early stage of epilepsy, and take personalized interventions, to improve patients' prognosis, reduce serious comorbidity, and save social resources. Herein, we developed and validated a nomogram prediction model for children with DRE. Methods: The training set was patients with epilepsy who visited the Children's Hospital of Soochow University (Suzhou Industrial Park, Jiangsu Province, China) between January 2015 and December 2017. The independent risk factors for DRE were screened by univariate and multivariate logistic regression analyses using SPSS21 software. The nomogram was designed according to the regression coefficient. The nomogram was validated in the training and validation sets. Internal validation was conducted using bootstrapping analyses. We also externally validated this instrument in patients with epilepsy from the Children's Hospital of Soochow University (Gusu District, Jiangsu Province, China) and Yancheng Maternal and Child Health Hospital between January 2018 and December 2018. The nomogram's performance was assessed by concordance (C-index), calibration curves, as well as GiViTI calibration belts. Results: Multivariate logistic regression analysis of 679 children with epilepsy from the Children's Hospital of Soochow University (Suzhou Industrial Park, Jiangsu Province, China) showed that onset age<1, status epilepticus (SE), focal seizure, > 20 pre-treatment seizures, clear etiology (caused by genetic, structural, metabolic, or infectious), development and epileptic encephalopathy (DEE), and neurological abnormalities were all independent risk factors for DRE. The AUC of 0.92 for the training set compared to that of 0.91 for the validation set suggested a good discrimination ability of the prediction model. The C-index was 0.92 and 0.91 in the training and validation sets. Additionally, both good calibration curves and GiViTI calibration belts (P-value: 0.849 and 0.291, respectively) demonstrated that the predicted risks had strong consistency with the observed outcomes, suggesting that the prediction model in both groups was perfectly calibrated. Conclusion: A nomogram prediction model for DRE was developed, with good discrimination and calibration in the training set and the validation set. Furthermore, the model demonstrated great accuracy, consistency, and prediction ability. Therefore, the nomogram prediction model can aid in the timely identification of DRE in children.

Use of perampanel in children with refractory epilepsy of genetic aetiology

OBJECTIVE: Pathogenic mutations in refractory childhood epilepsy are being increasingly discovered. In this study, we analysed the efficacy and tolerability of perampanel as treatment for genetically-related refractory childhood epilepsy. METHODS: This prospective study, conducted in China, included 50 patients with refractory epilepsy of genetic aetiology, who were treated with adjunctive perampanel therapy. Perampanel treatment was considered effective when the seizure frequency was reduced by >50%. Perampanel treatment was evaluated over at least nine months, from January 2020. RESULTS: A total of 184 paediatric patients with refractory epilepsy received addon perampanel therapy, and of these, 128 received treatment for ≥nine months and underwent genetic analysis. Fifty children were identified with pathogenic or likely pathogenic variants. A total of 24 different causative monogenic mutations were found, and the most common causative monogenic variants were observed in the SCN1A gene (n = 15). The mean maximal dose of perampanel was 3.4±1.2 mg/day in responders. The response rates to perampanel in children with genetically-related refractory epilepsy (n=50) were 68.0%, 58.0%, and 46.0% at three, six and nine months post-initiation, respectively. Adverse events were reported in 23 patients (46.0%) with genetic aetiology. Somnolence, ataxia, and irritability were the most common adverse events. The response rates to perampanel in children with pathogenic or likely pathogenic variants associated with Dravet syndrome, tuberous sclerosis, mitochondrial encephalopathy with lactic acidosis and stroke-like episodes, Rett syndrome, and dentatorubral-pallidoluysian atrophy were high. SIGNIFICANCE: A low maintenance dose of perampanel may be effective and well-tolerated as adjunctive treatment in children with refractory epilepsy of genetic aetiology.

CELSR3 variants are associated with febrile seizures and epilepsy with antecedent febrile seizures.

AIMS: To identify novel pathogenic gene of febrile seizures (FS)/epilepsy with antecedent FS (EFS+). METHODS: The trio-based whole-exome sequencing was performed in a cohort of 462 cases with FS/EFS+. Silico programs, sequence alignment, and protein modeling were used to predict the damaging of variants. Statistical testing was performed to analyze gene-based burden of variants. RESULTS: Five heterozygous missense variants in CELSR3 were detected in five cases (families) with eight individuals (five females, three males) affected. Two variants were de novo, and three were identified in families with more than one individual affected. All the variants were predicted to be damaging in silico tools. Protein modeling showed that the variants resulted in disappearance of multiple hydrogen bonds and one disulfide bond, which potentially caused functional impairments of protein. The frequency of CELSR3 variants identified in this study was significantly higher than that in controls. All affected individuals were diagnosed with FS/EFS+, including six patients with FS and two patients with EFS+. All cases presented favorable outcomes without neurodevelopmental disorders. CONCLUSIONS: CELSR3 variants are potentially associated with FS/EFS+.

Effectiveness and safety of perampanel in Chinese paediatric patients (2-14 years) with refractory epilepsy: a retrospective, observational study.

We report our clinical experience of the effectiveness and tolerability of adjunctive perampanel treatment in a Chinese paediatric population with refractory epilepsy. We also compare the effectiveness and tolerability of perampanel with or without concomitant oxcarbazepine or levetiracetam. This retrospective observational study was conducted from September 2019 to September 2020 in the paediatric neurology clinics of two tertiary hospitals in the Chinese mainland. We reviewed the data obtained from 96 paediatric patients aged 2-14 years whose seizures were pharmacoresistant and who could be followed up for a minimum of six months. The effectiveness was estimated by the perampanel response rate at 6- and 12-month follow-up evaluations. Adverse events were also recorded. Patients were stratified by age (2-7 and 7-14 years), and with/without concomitant oxcarbazepine or levetiracetam. The population comprised 96 patients with refractory epilepsy. The retention rate was 84.4% and 81.0% at 6 and 12 months, respectively. The most common dose used was 4 mg (48.5%). Corresponding 50% responder and seizure freedom rates were as follows: 46.9% and 20.8% at six months, and 51.2% and 27.4% at 12 months, respectively. Adverse effects were reported in 22 patients (22.9%). The most common adverse effects were irritability, somnolence, and dizziness. The 50% and 100% responder rates and adverse effects were greater in patients aged 7-14 years than in those aged 2-7 years. The proportion of responder and seizure-free rates and adverse effects were similar with or without oxcarbazepine. Perampanel was more effective in patients concomitantly treated with levetiracetam, however, this did not result in significantly more adverse events, including aggression. The present study suggests that perampanel, with or without concomitant oxcarbazepine or levetiracetam, is generally safe, well-tolerated, and efficacious in paediatric patients with uncontrolled epilepsy in a clinical setting.

Trio exome sequencing identified a novel de novo WASF1 missense variant leading to recurrent site substitution in a Chinese patient with developmental delay, microcephaly, and early-onset seizures: A mutational hotspot p.Trp161 and literature review.

BACKGROUND: Neurodevelopmental disorder with absent language and variable seizures (NEDALVS, OMIM # 618707) is a newly described autosomal dominant condition caused by heterozygous de novo mutation in WASF1 gene. WASF1 is a key component of the WAVE regulatory complex (WRC) required for actin polymerization. So far, only 3 distinct truncating variants clustering at the WCA domain, 3 missense variants localized to the meander region and a copy number variant (CNV) of WASF1 have been identified among 11 NEDALVS cases previously reported. CASE REPORT: We report a pediatric patient carrying novel de novo heterozygous missense variant (NM_003931.2: c.481T > C, p.Trp161Arg) in WASF1 gene. During the first hospitalization at age of 5.5 months, the patient was initially diagnosed with infantile spasms, developmental delay (DD) and microcephaly due to nodding-like epileptic spasms in clusters and hypsarrhythmia on video-electroencephalography, lacking head control and body rollover, and abnormal head circumference 39 cm (<-2SD). The genetic diagnosis with a causal WASF1 variant detected by trio exome sequencing indicated the rare NEDALVS. LITERATURE REVIEW: All the reported NEDALVS cases published in the PubMed English literature were reviewed to summarize the genetic and phenotypic spectrum of this novel disorder. CONCLUSION: We describe the third patient with a recurrently mutated amino acid site at p.Trp161 in WASF1, currently the 12th patient with NEDALVS. This hotspot missense variant and the truncating variants in WASF1 lead to similar phenotypic patterns with core features of severe DD/ID, and seizures, hypotonia, and microcephaly frequently observed. Our finding expands the WASF1 mutation spectrum and confirms the de novo hotspot missense variant at p.Trp161, further supporting the association of the novel NEDALVS with WASF1 gene and the actin regulatory pathway.

A De Novo SEMA6B Variant in a Chinese Patient with Progressive Myoclonic Epilepsy-11 and Review of the Literature.

Progressive myoclonic epilepsy is a group of neurodegenerative diseases with complex clinical and genetic heterogeneity, which is associated with spontaneous or action-induced myoclonus and progressive neurodegeneration. Since 2020, 4 families with progressive myoclonic epilepsy-11 [OMIM#618876] have been reported with a very limited spectrum of SEMA6B pathogenic variants. In our study, whole-exome sequencing was used in a proband from a nonconsanguineous Chinese family presenting with growth retardation and recurrent atonic seizures. A deletion mutation (c.1960_1978del, p.Leu654Argfs*25) in the last exon of SEMA6B was detected, which is a de novo variant and pathogenic. The new genetic evidence we reported here strengthened the gene-disease relationship, and the gene curation level between SEMA6B and progressive myoclonic epilepsy-11 became "strong" following the ClinGen SOP. Therefore, the results of this study broaden the mutation spectrum of SEMA6B in different ethnic groups and strengthen the gene-disease relationship between SEMA6B and progressive myoclonic epilepsy-11.

De novo ATP1A2 variants in two Chinese children with alternating hemiplegia of childhood upgraded the gene-disease relationship and variant classification: a case report.

BACKGROUND: ATP1A2 gene mutation has been indicated to cause alternating hemiplegia of childhood (AHC); however, limited evidence supports this relationship so far. CASE PRESENTATION: We reported two Chinese patients with de novo ATP1A2 variants (c.970G>A and c.889G>A). Both patients presented with episodes of alternating hemiplegia, seizures and mild developmental delay. Brain magnetic resonance imaging revealed abnormal signals in both patients. CONCLUSIONS: The new genetic evidence we reported here strengthened the gene-disease relationship, and the gene curation level between ATP1A2 and AHC became "Moderate" following the ClinGen Standard Operation Procedure. Consequently, the two variants can be reclassified as likely pathogenic.

Systematic elucidation of the pharmacological mechanisms of Rhynchophylline for treating epilepsy via network pharmacology.

ABSTACT: BACKGROUND: Epilepsy, one of the most common neurological disorders, affects over 70 million people worldwide. Rhynchophylline displays a wide variety of pharmacologic actives. However, the pharmacologic effects of rhynchophylline and its mechanisms against epilepsy have not been systematically elucidated. METHODS: The oral bioavailability and druglikeness of rhynchophylline were evaluated using the Traditional Chinese Medicine Systems Pharmacology Database. Rhynchophylline target genes to treat epilepsy were identified using PharmMapper, SwissTargetPrediction and DrugBank databases integration. Protein-protein interaction analysis was carried out by utilizing the GeneMANIA database. WebGestalt was employed to perform Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. The drug-disease-target-Gene Ontology-pathway network was constructed using Cytoscape. RESULTS: The oral bioavailability and druglikeness of rhynchophylline were calculated to be 41.82% and 0.57, respectively. A total of 20 rhynchophylline target genes related to epilepsy were chosen. Among the 20 genes and their interacting genes, 54.00% shared protein domains and 16.61% displayed co-expression characteristics. Gene ontology, Kyoto Encyclopedia of Genes and Genomes and network analyses illustrate that these targets were significantly enriched in regulation of sensory perception, morphine addiction, neuroactive ligand-receptor interaction and other pathways or biological processes. CONCLUSION: In short, rhynchophylline targets multiple genes or proteins, biological processes and pathways. It shapes a multiple-layer network that exerts systematic pharmacologic activities on epilepsy.

Proteinuria as a presenting sign of combined methylmalonic acidemia and homocysteinemia: case report.

BACKGROUND: Disorders of the metabolism and absorption of vitamin B12 can lead to decrease in activity of methionine synthetase and methylmalonate coenzyme A mutase (MMUT), which results in increased levels of methylmalonic acid and homocysteine in blood and urine. Often, combined methylmalonic acidemia (MMA) and homocysteinemia is misdiagnosed due to a lack of specific symptoms. The clinical manifestations are diverse, but proteinuria as the initial presentation is rare. CASE PRESENTATION: Two cases of MMA with homocysteinemia in children are reported. Proteinuria were a primary presenting symptom, followed by anemia and neurologic symptoms (frequent convulsions and unstable walking, respectively). Screening of amino acids and acyl carnitine in serum showed that the propionyl carnitine:acetylcarnitine ratio increased. Profiling of urinary organic acids by gas chromatography-mass spectrometry revealed high levels of methylmalonic acid. Homocysteine content in blood was increased. Comprehensive genetic analyses of peripheral blood-derived DNA demonstrated heterozygous variants of methylmalonic aciduria type C and homocystinuria (MMACHC) and amnionless (AMN) genes in our two patients, respectively. After active treatment, the clinical manifestations in Case 1 were relieved and urinary protein ceased to be observed; Case 2 had persistent proteinuria and was lost to follow-up. CONCLUSIONS: Analyses of the organic acids in blood and urine suggested MMA combined with homocysteinemia. In such diseases, reports of renal damage are uncommon and proteinuria as the initial presentation is rare. Molecular analysis indicated two different genetic causes. Although the pathologic mechanisms were related to vitamin B12, the severity and prognosis of renal lesions were different. Therefore, gene detection provides new insights into inherited metabolic diseases.

Clinical and Molecular Characteristics of Two Chinese Children with Infantile Sandhoff Disease and Review of the Literature.

Infantile Sandhoff disease is an autosomal recessive inherited disease primarily characterized by cherry red spots in the retina, muscle weakness, seizure, truncal hypotonia, hyperacusis, developmental delay and regression. The pathogenic genetic defects of the HEXB gene, which encodes the ß subunit of the hexosaminidase A (ɑß) and hexosaminidase B (ßß) enzymes, cause deficiency of both the Hex A and Hex B enzymes, resulting in the deposition of GM2 ganglion glycerides in the lysosomes of the central nervous system and somatic cells. The aim of this study was to discover disease-causing variants of the HEXB gene in two Chinese families through the use of exome sequencing. By characterizing three novel variants by molecular genetics, bioinformatics analysis, and three-dimensional structure modeling, we showed that all these novel variants influenced the protein structure. The results broaden the variant spectrum of HEXB in different ethnic groups. Furthermore, not all patients diagnosed with infantile Sandhoff disease had characteristic cranial imaging findings, which can only be used as supplementary information for diagnosis. The results of this study may contribute to clinical management, genetic counseling, and gene-targeted treatments for Sandhoff disease.

Myeloid-Related Protein 8/14 Participates in the Progression of Experimental Pneumococcal Meningitis by Augmentation of Inflammation.

It has been reported that myeloid-related protein 8/14 (MRP8/14) participates in the progression of inflammation after release from neutrophils and monocytes. This study aimed to clarify the mechanism(s) of the MRP8/14-augmented inflammatory response in mice with pneumococcal meningitis. Streptococcus pneumoniae (SP) meningitis was established by intracerebral injection of SP suspension. Balb/c mice were randomly divided into four groups and received the following injections: phosphate-buffer saline (PBS), MRP8/14 alone, SP alone, and SP plus MRP8/14. At 6 h, 24 h and 48 h postinfection, the clinical disease status was measured by the modified neurological severity score test, body weight loss and degree of cerebral edema; mice were anaesthetized, blood samples and brain samples were collected and brain inflammation was detected by haematoxylin and eosin (HE) staining; tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), C-reactive protein (CRP) and monocyte chemoattractant protein-1 (MCP-1) levels in serum and brain homogenates were assessed by an enzyme-linked immunosorbent assay (ELISA), and the mRNA levels of the above cytokines in brain homogenates were measured by polymerase chain reaction (PCR); and the expression of nuclear factor-kappa B (NF-κB) p65 in brain tissues was determined by immunohistochemical assay. In this study, we identified that MRP8/14 substantially augmented the SP-stimulated inflammatory response, aggravated clinical disease status and exacerbated SP-induced brain edema in a murine model of pneumococcal meningitis. Exogenous administration of MRP8/14 significantly enhanced mRNA and protein expression of the proinflammatory cytokines and chemokines TNF-α, CRP, IL-6 and MCP-1 in brain homogenates and serum from mice with pneumococcal meningitis, which may be related to the NF-κB signalling pathway. We further found that MRP8/14 strongly augmented SP-induced phosphorylation of NF-κB p65 in brain tissue slices from the same model. In conclusion, our results indicated that MRP8/14 augmented the inflammatory response in mice with pneumococcal meningitis and contributed to the development of disease, which was probably through NF-κB signalling pathway activation.

ATP7B Mutation Detection and Pathogenicity Analysis: One Atypical Case of Wilson's Disease with Adrenocortical Insufficiency.

Wilson's disease (WD) is an autosomal recessive disorder caused by defective function of the copper-transporting ATP7B protein. Symptoms are typically related to the brain and liver, while endocrinologic abnormalities are rare. Here, we reported a 12-year-old female patient that was initially presented with unusual skin darkening and low serum level of adrenocorticotropic hormone and diagnosed as having adrenocortical insufficiency. We further screened the mutation in ATP7B by direct DNA sequencing and found compound heterozygous mutations: a known pathogenic mutation in exon8:c.2333G>T (Arg778Leu) inherited from her mother and a variant in intron4:c.1707 + 5G>A inherited from her father. To explore the pathogenicity of the intronic variant, a minigene splicing assay was used to determine the effects of the splicing variant by analyzing reverse transcription PCR of ATP7B minigene transcript production. The result indicated that the c.1707 + 5G>A variant resulted in exon 4 skipping. We herein identified that 1707 + 5G>A intron 4 variant is a pathogenic mutation. Molecular genetic analysis and laboratory examination definitely confirmed the patient's condition as WD. Clinical status improved considerably after penicillamine treatment. Our results extended the mutation spectrum of ATP7B gene and highlighted the importance of molecular genetic analysis for the accurate diagnosis of atypical WD. WD may have diverse presentations and should be considered in children especially presenting with adrenocortical insufficiency as initial symptom, and this study highlights the importance of screening for hormone abnormal in WD.

Activation of the TLR2-mediated downstream signaling pathways NF-κB and MAPK is responsible for B7-H3-augmented inflammatory response during S. pneumoniae infection.

It has been reported that B7-H3, a costimulatory protein, participates in the development and progression of experimental pneumococcal meningitis by amplifying the TLR2-mediated inflammatory response. This study attempted to clarify the pathway(s) of TLR2 signaling involved in B7-H3-augmented inflammatory response during S. pneumoniae infection. Murine microglial cell line N9 cells and primary murine microglial cells were infected with S. pneumoniae alone or in combination with B7-H3. Although B7-H3 stimulation failed to further enhance S. pneumoniae-upregulated mRNA and protein expression of TLR2, it strongly augmented S. pneumoniae-induced phosphorylation of NF-κB p65, MAPK p38, and ERK1/2 in both N9 cells and primary microglial cells. Notably, B7-H3 itself did not activate NF-κB p65, MAPK p38, and ERK1/2. Furthermore, deactivation of NF-κB p65, MAPK p38, and ERK1/2 with their specific inhibitors significantly attenuated B7-H3-amplified proinflammatory cytokine and chemokine release from S. pneumoniae-infected microglial cells. Importantly, blockage of NF-κB p65, MAPK p38, or ERK1/2 in vivo substantially diminished B7-H3-augmented TNF-α levels in the brain of S. pneumoniae-infected mice. These results indicate that the activation of both NF-κB and MAPKs is predominantly responsible for B7-H3-augmented inflammatory response during S. pneumoniae infection.

Dysregulation of zinc/lipid metabolism­associated genes in the rat hippocampus and cerebral cortex in early adulthood following recurrent neonatal seizures.

Although it has been established that recurrent or prolonged clinical seizures during infancy may cause lifelong brain damage, the underlying molecular mechanism is still not well elucidated. The present study, to the best of our knowledge, is the first to investigate the expression of twenty zinc (Zn)/lipid metabolism­associated genes in the hippocampus and cerebral cortex of rats following recurrent neonatal seizures. In the current study, 6­day­old Sprague­Dawley rats were randomly divided into control (CONT) and recurrent neonatal seizure (RS) groups. On postnatal day 35 (P35), mossy fiber sprouting and gene expression were assessed by Timm staining and reverse transcription­quantitative polymerase chain reaction, respectively. Of the twenty genes investigated, seven were significantly downregulated, while four were significantly upregulated in the RS group compared with CONT rats, which was observed in the hippocampus but not in the cerebral cortex. Meanwhile, aberrant mossy fiber sprouting was observed in the supragranular region of the dentate gyrus and Cornu Ammonis 3 subfield of the hippocampus in the RS group. In addition, linear correlation analysis identified significant associations between the expression of certain genes in the hippocampus, which accounted for 40% of the total fifty­five gene pairs among the eleven regulated genes. However, only eight gene pairs in the cerebral cortex exhibited significant positive associations, which accounted for 14.5% of the total. The results of the present study indicated the importance of hippocampal Zn/lipid metabolism­associated genes in recurrent neonatal seizure­induced aberrant mossy fiber sprouting, which may aid the identification of novel potential targets during epileptogenesis.

B7-H3 Augments Inflammatory Responses and Exacerbates Brain Damage via Amplifying NF-κB p65 and MAPK p38 Activation during Experimental Pneumococcal Meningitis.

The costimulatory protein B7-H3 has been shown to play a contributory role in the development and progression of experimental pneumococcal meningitis by augmentation of the innate immunity-associated inflammatory response via a TLR2-dependent manner. This study aimed to clarify the component(s) of TLR2-mediated signal transduction pathways responsible for B7-H3-augmented inflammatory response and subsequent brain damage during experimental pneumococcal meningitis. Administration of B7-H3 did not augment expression of TLR2 and other TLR2 upstream components, but led to an enhanced formation of MyD88-IRAK immunocomplex in the brain of S. pneumoniae-infected mice. Furthermore, B7-H3 substantially augmented S. pneumoniae-induced activation of TLR2 downstream NF-κB p65 and MAPK p38 pathways in the brain of S. pneumoniae-infected mice. Notably, blockage of NF-κB p65 and/or MAPK p38 with their specific inhibitors strongly attenuated B7-H3-amplified inflammatory response with significantly reduced proinflammatory cytokine and chemokine production, and markedly ameliorated B7-H3-exacerbated disruption of blood-brain barrier and severity of disease status in S. pneumoniae-infected mice. These results indicate that targeting NF-κB p65 and/or MAPK p38 may represent a promising therapeutic option for amelioration of overwhelming inflammatory response-associated brain injury frequently observed during pneumococcal meningitis.