OBJECTIVE: To explore the clinical phenotype and genetic basis of a child with early onset neurodevelopmental disorder with involuntary movement (NEDIM). METHODS: A child who presented at Department of Neurology of Hunan Children's Hospital on October 8, 2020 was selected as the study subject. Clinical data of the child were collected. Genomic DNA was extracted from peripheral blood samples of the child and his parents. Whole exome sequencing (WES) was carried out for the child. Candidate variant was verified by Sanger sequencing and bioinformatic analysis. Relevant literature was searched from the CNKI, PubMed and Google Scholar databases to summarize the clinical phenotypes and genetic variants of the patients. RESULTS: This child was a 3-year-and-3-month boy with involuntary trembling of limbs and motor and language delay. WES revealed that the child has harbored a c.626G>A (p.Arg209His) variant of the GNAO1 gene. Sanger sequencing confirmed that neither of his parents has carried the same variant. The variant had been reported in HGMD and ClinVar databases, but not in the dbSNP, ExAC and 1000 Genomes databases. Prediction with SIFT, PolyPhen-2, and Mutation Taster online software suggested that the variant may be deleterious to the protein function. By UniProt database analysis, the encode amino acid is highly conserved among various species. Prediction with Modeller and PyMOL software indicated that the variant may affect the function of GαO protein. Based on the guideline of the American College of Medical Genetics and Genomics (ACMG), the variant was rated as pathogenic. CONCLUSION The GNAO1 gene c.626G>A (p.Arg209His) variant probably underlay the NEDIM in this child. Above finding has expanded the phenotypic spectrum of GNAO1 gene c.626G>A (p.Arg209His) variant and provided a reference for clinical diagnosis and genetic counseling.
Humans ; Computational Biology ; Genetic Counseling ; Genomics ; Mutation ; Neurodevelopmental Disorders/genetics* ; Dyskinesias ; GTP-Binding Protein alpha Subunits, Gi-Go

Proto-Oncogene Proteins c-akt ; TOR Serine-Threonine Kinases ; GTP-Binding Protein alpha Subunits, Gi-Go/metabolism*

The Cre/LoxP system is a well-established approach to spatially and temporally control genetic inactivation. The calcium/calmodulin-dependent protein kinase II alpha subunit (CaMKIIalpha) promoter limits expression to specific regions of the forebrain and thus has been utilized for the brain-specific inactivation of the genes. Here, we show that CaMKIIalpha-Cre can be utilized for simultaneous inactivation of genes in the adult brain and in male germ cells. Double transgenic Rosa26(+/stop-lacZ)::CaMKIIalpha-Cre(+/Cre) mice generated by crossing CaMKIIalpha-Cre(+/Cre) mice with floxed ROSA26 lacZ reporter (Rosa26(+/stop-lacZ)) mice exhibited lacZ expression in the brain and testis. When these mice were mated to wild-type females, about 27% of the offspring were whole body blue by X-gal staining without inheriting the Cre transgene. These results indicate that recombination can occur in the germ cells of male Rosa26(+/stop-lacZ)::CaMKIIalpha-Cre(+/Cre) mice. Similarly, when double transgenic Gnao(+/f)::CaMKIIalpha-Cre(+/Cre) mice carrying a floxed Go-alpha gene (Gnao(f/f)) were backcrossed to wild-type females, approximately 22% of the offspring carried the disrupted allele (Gnao(Delta)) without inheriting the Cre transgene. The Gnao(Delta/Delta) mice closely resembled conventional Go-alpha knockout mice (Gnao(-/-)) with respect to impairment of their behavior. Thus, we conclude that CaMKIIalpha-Cre mice afford recombination for both tissue- and time-controlled inactivation of floxed target genes in the brain and for their permanent disruption. This work also emphasizes that extra caution should be exercised in utilizing CaMKIIalpha-Cre mice as breeding pairs.
Animals ; Brain/*metabolism ; Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics ; Female ; GTP-Binding Protein alpha Subunits, Gi-Go/*genetics ; *Gene Deletion ; Gene Knockout Techniques/*methods ; Male ; Mice ; RNA, Untranslated/genetics ; Recombination, Genetic ; Spermatozoa/*metabolism

OBJECTIVE: To analyze the differentially expressed proteins in the dorsal raphe nucleus of rats treated with olanzapine and explore the possible mechanism of metabolic disorders in the early stage of olanzapine treatment. METHODS: Twenty male and 20 female SD rats were both randomized equally into olanzapine group and control group for daily treatment with olanzapine and saline for 4 weeks, respectively. One hour after the last treatment, the dorsal raphe nucleus of the rats was dissected for proteomic analysis using iTRAQ combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). GO, KEGG pathway, COG, pathways and protein interaction network analyses of the differentially expressed proteins were performed. Several target genes were selected from the proteomic list, and their expression levels in the dorsal raphe nucleus of another 24 mice with identical grouping and treatment using real time real-time quantitative PCR and Western blotting. RESULTS: A total of 214 differentially expressed proteins were identified in the dorsal raphe nucleus of olanzapine-treated mice, including 72 unregulated and 142 downregulated proteins. GO analyses showed that the differentially expressed proteins were enriched in cellular process, biological regulation, metabolic process, response to stimulus, multicellular organismal process, bindings, catalytic activity, molecular function regulator and transcription regulator activity. KEGG analysis suggested that these proteins were enriched in fluid shear stress and atherosclerosis, serotonergic synapse, butanoate metabolism, thyroid hormone synthesis and IL-17 signaling pathway. The differentially expressed proteins Cav1, Hsp90b1, Canx, Gnai1, MAPK9, and LOC685513 were located at the nodes of the protein-protein interaction network in close relation with metabolic disorders. In olanzapine-treated mice, the expression of Hmgcs2, a negative regulator of apoptosis, was significantly down-regulated in the dorsal raphe nucleus, where the expressions of Pla2g4e, Slc6a4 and Gnai1 involved in serotonergic synapse were significantly upregulated. CONCLUSION In the early stage of treatment, olanzapine may contribute to the occurrence of metabolic disorders in rats by regulating the expressions of Cav1, Hsp90b1, Canx, Gnai1, MAPK9, LOC685513 (Gng14) and 5-HTR2 synapse-related proteins in the dorsal raphe nucleus.
Animals ; Chromatography, Liquid ; Computational Biology ; Dorsal Raphe Nucleus ; Female ; GTP-Binding Protein alpha Subunits, Gi-Go ; Male ; Mice ; Olanzapine/adverse effects* ; Proteomics ; Rats ; Rats, Sprague-Dawley ; Tandem Mass Spectrometry

Helicobacter pylori (H. pylori) is an important risk factor for chronic gastritis, peptic ulcer, and gastric cancer. Proteinase-activated receptor 2 (PAR2), subgroup of G-protein coupled receptor family, is highly expressed in gastric cancer, and chronic expression of cyclooxygenase-2 (COX-2) plays an important role in H. pylori-associated gastric carcinogenesis and inflammation. We previously demonstrated that H. pylori induced the expression of PAR2 and COX-2 in gastric epithelial cells. Present study aims to investigate whether COX-2 expression induced by H. pylori in Korean isolates is mediated by PAR2 via activation of Gi protein and Src kinase in gastric epithelial AGS cells. Results showed that H. pylori-induced COX-2 expression was inhibited in the cells transfected with antisense oligonucleotide for PAR2 or treated with Gi protein blocker pertussis toxin, Src kinase inhibitor herbimycin A and soybean trypsin inbitor, indicating that COX-2 expression is mediated by PAR2 through activation of Gi protein and Src kinase in gastric epithelial cells infected with H. pylori in Korean isolates. Thus, targeting the activation of PAR2 may be beneficial for prevention or treatment of gastric inflammation and carcinogenesis associated with H. pylori infection.
Benzoquinones/*pharmacology ; Cell Line, Tumor ; Cyclooxygenase 2/genetics/*metabolism ; Epithelial Cells/enzymology/metabolism/microbiology ; GTP-Binding Protein alpha Subunits, Gi-Go/metabolism ; Gastric Mucosa/enzymology/metabolism/*microbiology ; *Helicobacter pylori ; Humans ; Lactams, Macrocyclic/*pharmacology ; Oligonucleotides, Antisense ; Pertussis Toxin/*pharmacology ; RNA, Messenger/metabolism ; Receptor, PAR-2/*physiology ; src-Family Kinases/metabolism

Lysophosphatidic acid (LPA) is a bioactive phospholipids and involves in various cellular events, including tumor cell migration. In the present study, we investigated LPA receptor and its transactivation to EGFR for cyclooxygenase-2 (COX-2) expression and cell migration in CAOV-3 ovarian cancer cells. LPA induced COX-2 expression in a dose-dependent manner, and pretreatment of the cells with pharmacological inhibitors of Gi (pertussis toxin), Src (PP2), EGF receptor (EGFR) (AG1478), ERK (PD98059) significantly inhibited LPA- induced COX-2 expression. Consistent to these results, transfection of the cells with selective Src siRNA attenuated COX-2 expression by LPA. LPA stimulated CAOV-3 cell migration that was abrogated by pharmacological inhibitors and antibody of EP2. Higher expression of LPA2 mRNA was observed in CAOV-3 cells, and transfection of the cells with a selective LPA2 siRNA significantly inhibited LPA-induced activation of EGFR and ERK, as well as COX-2 expression. Importantly, LPA2 siRNA also blocked LPA-induced ovarian cancer cell migration. Collectively, our results clearly show the significance of LPA2 and Gi/Src pathway for LPA-induced COX-2 expression and cell migration that could be a promising drug target for ovarian cancer cell metastasis.
Butadienes/pharmacology ; Cell Line, Tumor ; Cell Movement/drug effects/*physiology ; Cyclooxygenase 2/*biosynthesis ; Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors/metabolism ; Female ; Flavonoids/pharmacology ; GTP-Binding Protein alpha Subunits, Gi-Go/antagonists & inhibitors/*metabolism ; Humans ; Lysophospholipids/pharmacology ; Nitriles/pharmacology ; Ovarian Neoplasms/metabolism/*pathology ; Pertussis Toxin/pharmacology ; Protein-Tyrosine Kinases/antagonists & inhibitors/*metabolism ; Proto-Oncogene Proteins/antagonists & inhibitors/*metabolism ; Pyrimidines/pharmacology ; Receptor, Epidermal Growth Factor/antagonists & inhibitors/metabolism ; Receptors, Lysophosphatidic Acid/*metabolism ; Receptors, Prostaglandin E/metabolism ; Signal Transduction ; Transcriptional Activation ; Tyrphostins/pharmacology