Focal cortical dysplasia II caused by brain somatic mutation of IRS-1 is associated with ERK signaling pathway activation.

Somatic mutations have been identified in 10% to 63% of focal cortical dysplasia type II samples, primarily linked to the mTOR pathway. When the causative genetic mutations are not identified, this opens the possibility of discovering new pathogenic genes or pathways that could be contributing to the condition. In our previous study, we identified a novel candidate pathogenic somatic variant of IRS-1 c.1791dupG in the brain tissue of a child with focal cortical dysplasia type II. This study further explored the variant's role in causing type II focal cortical dysplasia through in vitro overexpression in 293T and SH-SY5Y cells and in vivo evaluation via in utero electroporation in fetal brains, assessing effects on neuronal migration, morphology, and network integrity. It was found that the mutant IRS-1 variant led to hyperactivity of p-ERK, increased cell volume, and was predominantly associated with the MAPK signaling pathway. In vivo, the IRS-1 c.1791dupG variant induced abnormal neuron migration, cytomegaly, and network hyperexcitability. Notably, the ERK inhibitor GDC-0994, rather than the mTOR inhibitor rapamycin, effectively rescued the neuronal defects. This study directly highlighted the ERK signaling pathway's role in the pathogenesis of focal cortical dysplasia II and provided a new therapeutic target for cases of focal cortical dysplasia II that are not treatable by rapamycin analogs.

Neurological function and drug-refractory epilepsy in Sturge-Weber syndrome children: a retrospective analysis.

Epilepsy in Sturge-Weber syndrome (SWS) is common, but drug-refractory epilepsy (DRE) in SWS has rarely been studied in children. We investigated the characteristics of epilepsy and risk factors for DRE in children with SWS. A retrospective study was conducted to analyze the clinical characteristics of children with SWS with epilepsy in our hospital from January 2013 to October 2022. Univariate and multivariate logistic analyses were performed to investigate the factors influencing DRE in children with SWS. A total of 35 SWS children with epilepsy were included (51% male; mean age of presentation 3.6 ± 0.5 years), 71% of children with SWS had their first seizure within the first year of life, and the most common type of seizure was focal seizure (77%). Eleven (31%) patients developed DRE. The median age of onset for the first seizure was 1.0 years and all these cases were of SWS type I. Multivariate logistic analysis revealed that stroke-like episodes and seizure clusters were risk factors for DRE in SWS children. A poor neurological function group was observed in twenty-five children with SWS. Status epilepticus was a risk factor that affected the neurological function of SWS children with epilepsy.  Conclusion: The study explored the epileptic features of children with SWS. The results revealed that stroke-like episodes and seizure clusters are risk factors for DRE in children with SWS. The occurrence of status epilepticus impacts the neurological function of SWS children with epilepsy. Thus, long-term follow-up is necessary to monitor outcomes. What is Known: • Sturge-Weber syndrome (SWS) is a rare neurocutaneous disorder, over 75% of children with SWS experience seizures, and 30-57% develop drug-refractory epilepsy (DRE), which leads to a poor outcome. • Drug-refractory epilepsy in SWS has been rarely studied in children, and the risk factors associated with DRE are unclear. What is New: • Clinical features of SWS children with drug-refractory epilepsy. • In SWS, stroke-like episodes and seizure clusters are risk factors of DRE, the occurrence of status epilepticus impacts the neurological function.

In Vitro Effects of Acitretin on Human Neuronal SH-SY5Y Cells.

Acitretin is an oral drug approved by the Food and Drug Administration that is commonly used to treat psoriasis. In recent years, acitretin has been identified as a candidate drug for the treatment of Alzheimer's disease, but its role in neuronal development is still unclear. In this study, the human neuroblastoma cell line SH-SY5Y was used as a model to study neuronal differentiation. We found that acitretin effectively promoted the differentiation of SH-SY5Y cells into neuronal cells and upregulated the expression of the neuronal marker ß-III tubulin and the mature neuronal marker NFH. Differentially expressed genes were identified by RNA sequencing and analyzed by bioinformatics approaches. The results showed that genes associated with neuron development-related pathways, such as SSPO and KCNT1, had significant changes in expression. Analysis showed that PRKCA and CAMK2B may play important roles in the process by which acitretin promotes neurodevelopment. Through whole-cell patch clamping and a microelectrode array assay, we found that acitretin-treated neurons generated electrical spikes similar to those generated by mature neurons. This study provided evidence to support an accessible and safe model of neuron-like cells and verified that acitretin can promote the differentiation of neurons and has the potential to treat brain tumors and neurodevelopmental and neurodegenerative diseases.

Neuroblastoma SH-SY5Y Cell Differentiation to Mature Neuron by AM580 Treatment.

Neuroblastoma is a type of developmental childhood cancer that arises from the neural crest. It is the most common pediatric solid tumor in the world. AM580 is a powerful cyto-differentiating molecule on acute promyelocytic leukemia cells and induced pluripotent stem cells, but its effect on neuroblastoma is still unknown. In this study, the neuronal differentiation impact of AM580 was investigated using the human neuroblastoma cell line SH-SY5Y as a model. AM580 successfully stimulated the SH-SY5Y cells to develop into neuron-like cells. Functional enrichment analysis of RNAseq data revealed that differentially expressed genes (DEGs) were substantially enriched for GO keywords and KEGG pathways linked to neuron development. Some potassium ion channel genes associated with neuronal excitation, such as KCNT1, were shown to be upregulated. Through the MEA tests, we found the AM580-induced neurons possessed electrical spikes as mature neurons. AM580 also induced the neuronal marker ß-tubulin III and mature neurons marker Neurofilament H. Our study proved that AM580 can promote the differentiation of neurons and has the potential to treat neuroblastoma, neurodevelopmental and neurodegenerative diseases.

Genetic analysis and identification of novel variations in Chinese patients with pediatric epilepsy by whole-exome sequencing.

OBJECTIVES: We aimed to investigate the genetic etiology of epilepsy in children, and to analyze the nature of genetic variation, the function of related genes, and the genotype-phenotype relationship. Moreover, the impact of the genetic diagnosis on prognosis and prenatal diagnosis will be discussed. METHODS: We recruited 218 pediatric epilepsy patients with onset ages ranging from postnatal 5 days to 3 years during a three-year collection period. WES was conducted only for the probands to screen for possible candidate genes. RESULTS: A total of 55 patients (25.2%) had positive genetic diagnoses. Autosomal dominant gene variants were the most common (34/55; 61.8%) and de novo variants (31/34; 91.2%) consistent with an autosomal dominant mode of inheritance. Among 64 variants identified in 35 genes, 33 (51.6%) were novel, previously unreported. Ion channel genes play critical roles in the pathogenesis of epilepsy, accounting for 58.8% (20/34) of the variants. A total of 31 (56.4%) families chose to have a prenatal diagnosis in subsequent pregnancies based on the genetic diagnosis. CONCLUSION: Our data suggest that applying WES in patients with epilepsy of unknown etiology can improve counseling and management. Early establishment of genetic diagnosis was necessary for counseling on recurrence risk and prenatal diagnosis. A large number of unreported variants were detected, widening the known spectrum of genetic variation related to epilepsy risk.

A novel NAPB splicing mutation identified by Trio-based exome sequencing is associated with early-onset epileptic encephalopathy.

N-ethylmaleimide-sensitive factor attachment proteins (NAP: NAPA and NAPB) play a role in Soluble N-ethylmaleimide-sensitive accessory protein receptor (SNARE) complex dissociation and recycling, associated with neuronal regulation and brain development and various severe early-onset epilepsies. Here, we report two patients from a Chinese family presenting with unexplained early-onset epileptic encephalopathies (EOEE) syndrome characterized by multifocal seizures, profound intellectual disability and global developmental delay. We identified the homozygous c.433-1G > A variant of the NAPB as the causative by trio-based exome sequencing. The novel splicing mutation in NAPB was third variant reported associated with EOEE syndrome. Our results gave further hints on the associations of variants in NAPB with EOEE and indicated that for patients with unexplained EOEE, the NAPB gene should be included into the data analysis from whole exome sequencing, which contributes to uncover more patients affected and rich the phenotypic spectrum.

The novel circCLK3/miR-320a/FoxM1 axis promotes cervical cancer progression.

As a new class of non-coding RNA, circular RNAs (circRNAs) play crucial roles in the development and progression of various cancers. However, the detailed functions of circRNAs in cervical cancer have seldom been reported. In this study, circRNA sequence was applied to detect the differentially expressed circRNAs between cervical cancer tissues and adjacent normal tissues. The relationships between circCLK3 level with clinicopathological characteristics and prognosis were analyzed. In vitro CCK-8, cell count, cell colony, cell wound healing, transwell migration and invasion, and in vivo tumorigenesis and lung metastasis models were performed to evaluate the functions of circCLK3. The pull-down, RNA immunoprecipitation (RIP), luciferase reporter and rescue assays were employed to clarify the interaction between circCLK3 and miR-320a and the regulation of miR-320a on FoxM1. We found that the level of circCLK3 was remarkably higher in cervical cancer tissues than in adjacent normal tissues, and closely associated with tumor differentiation, FIGO stage and depth of stromal invasion. Down-regulated circCLK3 evidently inhibited cell growth and metastasis of cervical cancer in vitro and in vivo, while up-regulated circCLK3 significantly promoted cell growth and metastasis in vitro and in vivo. The pull-down, luciferase reporter and RIP assays demonstrated that circCLK3 directly bound to and sponge miR-320a. MiR-320a suppressed the expression of FoxM1 through directly binding to 3'UTR of FoxM1 mRNA. In addition, FoxM1 promoted cell proliferation, migration, and invasion of cervical cancer, while miR-320a suppressed cell proliferation, migration, and invasion through suppressing FoxM1, and circCLK3 enhanced cell proliferation, migration and invasion through sponging miR-320a and promoting FoxM1 expression. In summary, circCLK3 may serve as a novel diagnostic biomarker for disease progression and a promising molecular target for early diagnoses and treatments of cervical cancer.

Development of CRISPR-Mediated Systems in the Study of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a severe type of X-linked recessive degenerative muscle disease caused by mutations in the dystrophin (DMD) gene on the X chromosome. The DMD gene is complex, consisting of 79 exons, and mutations cause changes in the DMD mRNA so that the reading frame is altered, and the muscle-specific isoform of the dystrophin protein is either absent or truncated with variable residual function. The emerging CRISPR-Cas9-mediated genome editing technique is being developed as a potential therapeutic approach to treat DMD because it can permanently replace the mutated dystrophin gene with the normal gene. Prenatal DNA testing can inform whether the female fetus is a carrier of DMD, and the male fetus has inherited a mutation from his mother (50% chance of both). This article summarizes the present status of current and future treatments for DMD.

[Genetic testing of chorionic villi from abortuses during early pregnancy].

OBJECTIVE: To explore the prevalence and characteristics of chromosomal abnormalities in abortuses during early pregnancy with single nucleotide polymorphism microarray (SNP-array). METHODS: For 520 abortuses, copy number variations (CNVs) in chorionic villi were analyzed with SNP-array. RESULTS: In 510 (98.1%) of the samples, the analysis was successful. Among these, 57.6% (294/510) of the samples were found to harbor clinically significant chromosomal abnormalities. 38.8% of the samples (198/510) had a normal result. 2.4% (12/510) of the samples harbored benign CNVs, and 1.2% (6/510) harbored variants of uncertain significance (VOUS). Aneuploidies, polyploidies, pathogenic CNVs and uniparental disomies (UPD) had accounted for 75.2% (221/294), 13.9% (41/294), 8.2% (24/294), and 2.7% (8/294) of the samples, respectively. 45,XO was the most common finding, which was followed by trisomy 16 and trisomy 22. 69,XXY was the most common polyploidy. CONCLUSION: Chromosomal abnormalities are the main cause for early miscarriage, among which aneuploidies are most common. The prevalence of aneuploidies is significantly increased among women over 35. SNP-array analysis has the advantage of high success rate, high resolution and great accuracy, but the clinical significance of microdeletions/microduplications found by SNP-array can be difficult for interpretation.

Exploring the cause of early miscarriage with SNP-array analysis and karyotyping.

OBJECTIVE: The aim of this study is to explore the cause of miscarriage, providing risk assessment to guide the next pregnancy. METHODS: Four hundred eighty-four products-of-conception (POC) samples were analyzed by single nucleotide polymorphism (SNP) array, and peripheral blood samples of couples were collected for karyotyping or fluorescence in situ hybridization (FISH) analysis. RESULTS: Four hundred sixty-eight of the 484 (96.7%) fresh POC samples were successfully analyzed using SNP-array. The rate of clinically significant chromosomal abnormalities were 58.3% (274/468), in which rates of aneuploidy, polyploidy, partial aneuploidy, uniparental isodisomy (isoUPD), and pathogenic microdeletion/microduplication were 43.4% (203/468), 8.8% (41/468), 3.6% (17/468), 1.9% (9/48), and 0.9% (4/468), respectively. The percentage of embryonic chromosomal abnormalities significantly increased with maternal age of patients older than 35 years old. Among 468 couples, 12 major chromosomal rearrangements were detected by G-banding, including nine reciprocal translocations, two Robertsonian translocations, and one superfemale. CONCLUSIONS: Chromosome abnormality is the main causes of early miscarriage, and aneuploidies are the most common type of chromosomal abnormalities. Application of SNP array and karyotyping in early miscarriage can provide more genetic information about miscarriage, providing risk assessment to guide the next pregnancy.

Generation of ZZUi008-A, a transgene-free, induced pluripotent stem cell line derived from chorionic villi cells of a fetus with Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a common X-linked recessive disorder for which there is no present cure. In this paper, we reported the generation of ZZUi008-A, an induced pluripotent stem cell(iPSC) line derived from chorionic villus(CV) cells of a fetus with a deletion mutation in exon 33 of the dystrophin gene (DMD). The cell line was generated using feeder-free and virus-free conditions, and the established cell line retains the original DMD mutation, a normal karyotype, expresses pluripotency markers, able to differentiate into three lineages. This ZZUi008-A cell line could provide a promising tool to study this complex disease.

[IDUA gene mutation analysis and prenatal diagnosis of two families affected with mucopolysaccharidosis type I].

OBJECTIVE: To analyze mutations of IDUA gene in two pedigrees affected with mucopolysaccharidosis type I and provide prenatal diagnosis for them. METHODS: The 14 exons of the IDUA gene were subjected to PCR amplification and Sanger sequencing. RESULTS: For pedigree 1, the proband was found to harbor compound heterozygous mutations c.46-57delTCGCTCCTGGCC (p.Ser16_Ala19del) of exon 1 and c.1147delC (p.Arg383Alafs*57) of exon 8 of the IDUA gene, which were inherited from his father and mother, respectively. The latter was unreported previously. Prenatal diagnosis suggested that the fetus has carried a heterozygous c.46-57delTCGCTCCTGGCC mutation. For family 2, the proband was also found to carry compound mutations of the IDUA gene, namely c.721T to C (p.Cys241Arg) of exon 6 and c.1491delG (p.Thr497fs27) of exon 8, which were inherited from her mother and father, respectively. Neither mutation was reported previously. Prenatal diagnosis suggested that the fetus has carried a heterozygous c.721T to C mutation. CONCLUSION: Mutations of the IDUA gene probably underlie the MPS-I in both pedigrees. Above results have enriched the spectrum of IDUA gene mutations and facilitated prenatal diagnosis for both families.