[Clinical and genetic analyses of Joubert syndrome in children]. / 儿童Joubertç»¼åˆå¾ä¸´åºŠåŠé—ä¼ å­¦åˆ†æž.

OBJECTIVES: To study the clinical and genetic features of Joubert syndrome (JS) in children. METHODS: A retrospective analysis was performed on the clinical data, genetic data, and follow-up data of 20 children who were diagnosed with JS in the Department of Children's Rehabilitation, the Third Affiliated Hospital of Zhengzhou University, from January 2017 to July 2022. RESULTS: Among the 20 children with JS, there were 11 boys and 9 girls. The common clinical manifestations were developmental delay (20 children, 100%), abnormal eye movement (19 children, 95%), and hypotonia (16 children, 80%), followed by abnormal respiratory rhythm in 5 children (25%) and unusual facies (including prominent forehead, low-set ears, and triangular mouth) in 3 children (15%), and no limb deformity was observed. All 20 children (100%) had the typical "molar tooth sign" and "midline cleft syndrome" on head images, and 6 children (30%) had abnormal eye examination results. Genetic testing was performed on 7 children and revealed 6 pathogenic genes, i.e., the CPLANE1, RPGRIP1L, MKS1, CC2D2A, CEP120, and AHI1 genes. CONCLUSIONS: For children with developmental delay, especially those with abnormal eye movement and hypotonia, it is recommended to perform a head imaging examination to determine the presence or absence of "molar tooth sign" and "midline cleft syndrome", so as to screen for JS to avoid missed diagnosis and misdiagnosis. There are many pathogenic genes for JS, and whole-exome sequencing can assist in the diagnosis of JS.

[Analysis of NOVA2 gene variant in a child with Neurodevelopmental disorder with or without autistic features and/or structural brain abnormalities].

OBJECTIVE: To explore the genetic basis for a child with Neurodevelopmental disorder with or without autistic features and/or structural brain abnormalities (NEDASB). METHODS: A child with NEDASB who presented at the Third Affiliated Hospital of Zhengzhou University in July 2021 was selected as the subject. Peripheral blood samples of the child and her parents were collected and subjected to high-throughput sequencing. Candidate variant was verified by Sanger sequencing and bioinformatic analysis. RESULTS: The child was found to harbor a heterozygous c.820_828delinsCTTCA (p.Thr274Leufs*121) variant of the NOVA2 gene, for which both of her parents were of wild type. The variant was predicted as pathogenic based on the guidelines from the American College of Medical Genetics and Genomics. CONCLUSION: The heterozygous c.820_828delinsCTTCA (p.Thr274Leufs*121) variant of the NOVA2 gene probably underlay the disease in this child. Above finding has enriched the spectrum of NOVA2 gene variants and provided a basis for genetic counseling and prenatal diagnosis for this family.

Identification of a 5 bp duplicate in the AP1S2 gene of an individual with X-linked intellectual disability.

Adaptor-related protein complex 1 subunit sigma 2 (AP1S2) is a subunit of AP1 that is crucial for the reformation of the synaptic vesicle. Variants in AP1S2 have been reported to cause a rare neurodevelopmental disorder, Pettigrew syndrome (PGS) (OMIM: 304,340), which is characterized by walking delay, abnormal speech, mild to profound X-linked intellectual disability (XLID), and abnormal brain, and behaviors. Here, we describe a 2-year- and 5-month-old male patient who presented with global developmental delay (GDD). Trio whole exome sequencing (WES) revealed a 5 bp duplicate in the AP1S2 gene (NM_003916.5: exon 2: c.96_100dup, p. Leu34Glnfs*8) predicted to cause early termination of translation, which was inherited from the unaffected mother. The clinical features of our patient were consistent with previous reports. This is the second case in the Chinese family and the eleventh variant found in AP1S2-related XLID. Our findings expand the AP1S2 variant spectrum in neurodevelopmental disorders and provide evidence for the application of WES in PGS diagnosis.

High expression of cluster of differentiation 276 indicates poor prognosis in glioma.

BACKGROUND: Glioma is the central nervous system tumor with the highest incidence rate and the molecular detection of gliomas has been the focus of research. This study aimed to investigate the guiding effect of cluster of differentiation 276 (CD276) expression on the clinical prognosis of glioma. METHODS: The TCGA and CGGA databases were used to study whether CD 276 can be used as an independent prognostic factor for gliomas. Immunohistochemistry was used to detect the expression of CD276, isocitrate dehydrogenase-1 (IDH1), matrix metallopeptidase 9 (MMP9), p53, and Ki-67, and 1p/19q co-deletion was detected by fluorescence in situ hybridization (FISH). The effects of CD276 RNA interference (RNAi) on cell invasion, cell cycle and the expression of ß-catenin, tumor necrosis factor receptor 1 (TNFR1), and MMP9 were observed. Furthermore, the biological effects of CD276 gene knockout on intracranial transplanted tumors in nude mice were studied. RESULTS: CD276 expression was positively correlated with the extracellular matrix, collagen decomposition, and cell adhesion molecules. Immunohistochemistry and FISH showed that CD276 expression positively correlated with the glioma grade, p53 mutation, Ki-67 proliferation, and MMP9 expression; however, it negatively correlated with IDH1 mutation, 1p/19q co-deletion, and the survival rate. CD276 RNAi in U87 cells inhibited cell proliferation, migration, and invasion, but had no effect on the cell cycle. CD276 inhibited the expression of ß-catenin, TNFR1, and MMP9 in U87 cells at the mRNA and protein levels. In vivo experiments showed that the tumor formation and invasion of the CD276 small interfering RNA glioma cell line in nude mice were reduced and the survival time was prolonged. CONCLUSIONS: The present study demonstrated that high expression of CD276 in gliomas indicates a poor prognosis.

Mutations disrupting neuritogenesis genes confer risk for cerebral palsy.

In addition to commonly associated environmental factors, genomic factors may cause cerebral palsy. We performed whole-exome sequencing of 250 parent-offspring trios, and observed enrichment of damaging de novo mutations in cerebral palsy cases. Eight genes had multiple damaging de novo mutations; of these, two (TUBA1A and CTNNB1) met genome-wide significance. We identified two novel monogenic etiologies, FBXO31 and RHOB, and showed that the RHOB mutation enhances active-state Rho effector binding while the FBXO31 mutation diminishes cyclin D levels. Candidate cerebral palsy risk genes overlapped with neurodevelopmental disorder genes. Network analyses identified enrichment of Rho GTPase, extracellular matrix, focal adhesion and cytoskeleton pathways. Cerebral palsy risk genes in enriched pathways were shown to regulate neuromotor function in a Drosophila reverse genetics screen. We estimate that 14% of cases could be attributed to an excess of damaging de novo or recessive variants. These findings provide evidence for genetically mediated dysregulation of early neuronal connectivity in cerebral palsy.

MicroRNA Let-7f-5p Promotes Bone Marrow Mesenchymal Stem Cells Survival by Targeting Caspase-3 in Alzheimer Disease Model.

Widespread death of transplanted mesenchymal stem cells (MSCs) hampers the development of stem cell therapy for Alzheimer disease (AD). Cell pre-conditioning might help cope with this challenge. We tested whether let-7f-5p-modified MSCs could prolong the survival of MSCs after transplantation. When exposed to Aß25-35in vitro, MSCs showed significant early apoptosis with decrease in the let-7f-5p levels and increased caspase-3 expression. Upregulating microRNA let-7f-5p in MSCs alleviated Aß25-35-induced apoptosis by decreasing the caspase-3 levels. After computerized analysis and the luciferase reporter assay, we identified that caspases-3 was the target gene of let-7f-5p. In vivo, hematoxylin and eosin staining confirmed the success of MSCs transplantation into the lateral ventricles, and the let-7f-5p upregulation group showed the lowest apoptotic rate of MSCs detected by TUNEL immunohistochemistry analysis and immunofluorescence. Similarly, bioluminescent imaging showed that let-7f-5p upregulation moderately prolonged the retention of MSCs in brain. In summary, we identified the anti-apoptotic role of let-7f-5p in Aß25-35-induced cytotoxicity, as well as the protective effect of let-7f-5p on survival of grafted MSCs by targeting caspase-3 in AD models. These findings show a promising approach of microRNA-modified MSCs transplantation as a therapy for neurodegenerative diseases.

Role of Hypoxia-Inducible Factors 1α (HIF1α) in SH-SY5Y Cell Autophagy Induced by Oxygen-Glucose Deprivation.

BACKGROUND HIF-1α plays an important role in hypoxia-ischemia brain damage. Accumulating evidences demonstrates that HIF-1α can contribute to cell autophagy. Oxygen-glucose deprivation (OGD) is a commonly used ischemic model in vitro. Our study was performed to investigate the influences of HIF-1α on autophagy in SH-SY5Y cells under OGD treatment. MATERIAL AND METHODS An OGD model was constructed in SH-SY5Y cells. PI method and MTT assay were used to test cell death and viability, respectively. Western blot assay was used to estimate the protein levels of HIF-1α and LC3. Quantitative GFP-LC3 light microscopy autophagy assay was performed for SH-SY5Y cells. 2ME2 and siRNA-HIF-1α were applied to investigate the effects of HIF-1α-knockdown on LC3 expression. Additionally, 3-MA (autophagy inhibitor) and autophagy inducer rapamycin (Rapa) were used to investigate the effects of autophagy on cell survival under OGD condition. RESULTS Under OGD, the apoptosis of SH-SY5Ycells was increased while cell viability rate was decreased. The expression of HIF-1α was increased along with the advancement of OGD treatment and achieved the highest level at 24 h. However, inhibiting HIF-1α expression decreased the cell apoptosis and increased cell viability. LC3-II expression was gradually increased with the duration of OGD condition and knockdown of HIF-1α resulted in decreased expression of LC3. Inhibiting autophagy significantly enhanced the viability and reduced the apoptosis of SH-SY5Y cells, while enhancing autophagy showed the opposite effects. CONCLUSIONS Enhanced expression of HIF-1α may be related to autophagy activation in SH-SY5Y cells, thus contributing to ischemic/hypoxic brain damage.

MicroRNA-9 promotes the neuronal differentiation of rat bone marrow mesenchymal stem cells by activating autophagy.

MicroRNA-9 (miR-9) has been shown to promote the differentiation of bone marrow mesenchymal stem cells into neuronal cells, but the precise mechanism is unclear. Our previous study confirmed that increased autophagic activity improved the efficiency of neuronal differentiation in bone marrow mesenchymal stem cells. Accumulating evidence reveals that miRNAs adjust the autophagic pathways. This study used miR-9-1 lentiviral vector and miR-9-1 inhibitor to modulate the expression level of miR-9. Autophagic activity and neuronal differentiation were measured by the number of light chain-3 (LC3)-positive dots, the ratio of LC3-II/LC3, and the expression levels of the neuronal markers enolase and microtubule-associated protein 2. Results showed that LC3-positive dots, the ratio of LC3-II/LC3, and expression of neuron specific enolase and microtubule-associated protein 2 increased in the miR-9(+) group. The above results suggest that autophagic activity increased and bone marrow mesenchymal stem cells were prone to differentiate into neuronal cells when miR-9 was overexpressed, demonstrating that miR-9 can promote neuronal differentiation by increasing autophagic activity.

The association between sex-related interleukin-6 gene polymorphisms and the risk for cerebral palsy.

BACKGROUND: The relationship between genetic factors and the development of cerebral palsy (CP) has recently attracted much attention. Polymorphisms in the genes encoding proinflammatory cytokines have been shown to be associated with susceptibility to perinatal brain injury and development of CP. Interleukin-6 (IL-6) is a proinflammatory cytokine that plays a pivotal role in neonatal brain injury, but conflicting results have been reported regarding the association between IL-6 single nucleotide polymorphisms (SNPs) and CP. The purpose of this study was to analyze IL-6 gene polymorphisms and protein expression and to explore the role of IL-6 in the Chinese CP population. METHODS: A total of 753 healthy controls and 713 CP patients were studied to detect the presence of five SNPs (rs1800796, rs2069837, rs2066992, rs2069840, and rs10242595) in the IL-6 locus. Of these, 77 healthy controls and 87 CP patients were selected for measurement of plasma IL-6 by Luminex assay. The SHEsis program was used to analyze the genotyping data. For all comparisons; multiple testing on each individual SNP was corrected by the SNPSpD program. RESULTS: There were no differences in allele or genotype frequencies between the overall CP patients and controls among the five genetic polymorphisms. However, subgroup analysis found significant sex-related differences in allele and genotype frequencies. Differences were found between spastic CP and controls in males for rs2069837; between CP with periventricular leukomalacia and controls in males for rs1800796 and rs2066992; and between term CP and controls in males for rs2069837. Plasma IL-6 levels were higher in CP patients than in the controls, and this difference was more robust in full-term male spastic CP patients. Furthermore, the genotype has an effect on IL-6 synthesis. CONCLUSIONS: The influence of IL-6 gene polymorphisms on IL-6 synthesis and the susceptibility to CP is related to sex and gestational age.

Association of Interleukin 6 gene polymorphisms with genetic susceptibilities to spastic tetraplegia in males: a case-control study.

BACKGROUND: Cerebral palsy (CP) is a group of non-progressive motor impairment and permanent disorders causing limitation of activity and abnormal posture. It may be caused by infection (such as chorioamnionitis), asphyxia or multiple genetic factors. The Interleukin 6 gene (IL6) was suggested to be involved in the susceptibilities to CP risk as a kind of proinflammatory cytokine. OBJECTIVE: To explore the genetic association between the polymorphisms of the IL6 gene and CP in the Chinese population. METHODS: A total of 542 CP patients and 483 healthy control children were recruited in this study to detect five single nucleotide polymorphisms (rs1800796, rs2069837, rs2066992, rs2069840, and rs10242595) in the IL6 locus. Genotyping of SNPs was performed by the MassArray platform-based genotyping approach. The SHEsis program was applied to analyze the genotyping data. RESULTS: Of the five selected SNPs, no significant allelic and genotypic association was found between CP patients and controls. However, subgroup analysis found significant differences in allele frequencies between spastic tetraplegia in males compared with controls at rs1800796 (OR=1.39, P=0.033, P=0.099 after SNPSpD correction) and rs2069837 (OR=1.58, P=0.012, P=0.035 after SNPSpD correction). The frequencies of the C allele of rs1800796 and the A allele of rs2069837 were greater in males with spastic tetraplegia than in the controls. The two SNPs haplotype rs1800796 (G) - rs2069837 (G) were also associated with a decreased risk of spastic tetraplegia in males (OR=0.619, P=0.009, P=0.027 after Bonferroni correction). CONCLUSION: Genetic variation of the IL6 gene may influence susceptibility to spastic tetraplegia in males and its role in cerebral palsy deserves further evaluation in a large-scale and well-designed study.