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*

Humans ; Brain Neoplasms/metabolism* ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Glioma/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Signal Transduction ; ATPases Associated with Diverse Cellular Activities/metabolism* ; Mitochondrial Proteins/metabolism* ; GTP-Binding Protein alpha Subunits/metabolism*

OBJECTIVE: To explore the genetic etiology of a Chinese pedigree affected with pseudohypoparathyroidism. METHODS: Peripheral blood samples of the proband and his parents were collected and subjected to trio-whole exome sequencing (trio-WES). Candidate variants were verified among the pedigree and 50 randomly selected healthy individuals through analysis of restriction fragment length polymorphism. Short tandem repeat (STR) linkage analysis was used to verify the parental origin of the pathogenic variants. RESULTS: Trio-WES and Sanger sequencing showed that the proband and his mother had both harbored a c.121C>G (p.His41Asp) variant of the GNAS gene, which was not found in other family members and the 50 healthy controls. The variant was not found in international databases. Based on guidelines from the American College of Medical Genetics and Genomics, the variant was predicted to be likely pathogenic. CONCLUSION The novel c.121C>G variant of the GNAS gene probably underlay the disease in this pedigree. Above finding has enriched the spectrum of GNAS gene variants.
Female ; Humans ; Pedigree ; East Asian People ; Mothers ; Exome Sequencing ; Pseudohypoparathyroidism/genetics* ; Mutation ; China ; Chromogranins/genetics* ; GTP-Binding Protein alpha Subunits, Gs/genetics*

More than 85% of patients with uveal melanoma (UM) carry a GNAQ or GNA11 mutation at a hotspot codon (Q209) that encodes G protein α subunit q/11 polypeptides (Gα_q/11). GNAQ/11 relies on palmitoylation for membrane association and signal transduction. Despite the palmitoylation of GNAQ/11 was discovered long before, its implication in UM remains unclear. Here, results of palmitoylation-targeted mutagenesis and chemical interference approaches revealed that the loss of GNAQ/11 palmitoylation substantially affected tumor cell proliferation and survival in UM cells. Palmitoylation inhibition through the mutation of palmitoylation sites suppressed GNAQ/11^Q209L-induced malignant transformation in NIH3T3 cells. Importantly, the palmitoylation-deficient oncogenic GNAQ/11 failed to rescue the cell death initiated by the knock down of endogenous GNAQ/11 oncogenes in UM cells, which are much more dependent on Gα_q/11 signaling for cell survival and proliferation than other melanoma cells without GNAQ/11 mutations. Furthermore, the palmitoylation inhibitor, 2-bromopalmitate, also specifically disrupted Gα_q/11 downstream signaling by interfering with the MAPK pathway and BCL2 survival pathway in GNAQ/11-mutant UM cells and showed a notable synergistic effect when applied in combination with the BCL2 inhibitor, ABT-199, in vitro. The findings validate that GNAQ/11 palmitoylation plays a critical role in UM and may serve as a promising therapeutic target for GNAQ/11-driven UM.
Humans ; Mice ; Animals ; Lipoylation ; NIH 3T3 Cells ; Uveal Neoplasms/genetics* ; Melanoma/genetics* ; Cell Proliferation ; Proto-Oncogene Proteins c-bcl-2 ; GTP-Binding Protein alpha Subunits, Gq-G11/genetics*

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

OBJECTIVE: To develop a multiplex PCR method for a rapid detection of Y chromosome-specific sequences in patients with Turner syndrome. METHODS: Nine genes were selected from various regions of the Y chromosome for designing the primers, which included SRY, TBL1Y, TSPY on the short arm of the Y chromosome, DDX3Y, HSFY1, RPS4Y2 and CDY1 on the long arm of Y chromosome and SHOX in the short arm and SPRY3 in the long arm of the pseudoautosomal region (PAR) of X and Y chromosomes. A multiplex PCR method for the nine genes in Y chromosome was established and optimized. The sensitivity was tested by using different amounts of genomic DNA. A total of 36 patients with Turner syndrome and a patient with male dwarfism with karyotype of 46, X, +mar were examined by the multiplex PCR method for the existence of materials from the Y chromosome. RESULTS: The optimization results of the multiplex PCR reaction system (50 μL) showed that when the final concentration of upstream and downstream of each pair of primers was 0.1 μM, the multiplex PCR reaction of the 9 pairs of primers clearly amplified the target with the expected band size, and there was no non-specific amplification. The bands were clearly visible when the amount of genomic DNA in the multiple PCR reaction system was as low as 1 ng. By using the method, we have examined the 36 patients with Turner syndrome. One patient with Turner syndrome with karyotype of 45,X[40]/47XYY[21] amplified specific seven genes on Y chromosome, 35 patients with Turner syndrome amplified only two target genes SHOX and SPRY3, but not the other seven specific genes on the Y chromosome, which was in keeping with the clinical manifestations of such patients. CONCLUSION This study established a multiplex PCR reaction system with nine genes, which can quickly and accurately screen Y chromosome materials in patients with Turner syndrome. It has the advantages of low cost, simple operation, high specificity and rapid turn-around time, and can be used to detect Turner syndrome patients with Y chromosome material in time. The method has provided a diagnostic basis for preventive gonad resection to prevent malignant gonadal tumors.
Humans ; Male ; Turner Syndrome/genetics* ; Multiplex Polymerase Chain Reaction ; Y Chromosome ; Karyotyping ; DNA Primers ; DNA ; Chromosomes, Human, Y/genetics* ; Transducin/genetics* ; Minor Histocompatibility Antigens ; DEAD-box RNA Helicases/genetics*

Pseudohypoparayhyroidism (PHP) is a rare autosomal dominant or recessive genetic disorder characterized by low calcium, high phosphorus, and target organ resistance to parathyroid. The clinical characteristics and genetic features in 4 patients with Type Ib PHP in the Third Xiangya Hospital, Central South University, have been reviewed. All 4 patients had low calcium, high phosphorus, and parathyroid resistance. Among them, 2 patients had slightly elevated thyroid stimulating hormone and mild features of Albright's hereditary osteodystrophy, and one patient had hypokalemia. No guanine nucleotide-binding protein alpha-stimulating activity polypeptide 1 (GNAS) and gene variant associated with hypokalemia were identified using the whole exome sequencing. The results of the methylation-specific multiple ligation-dependent probe amplification showed that there were abnormal methylation of the upstream differentially methylated regions of GNAS in the 4 patients. There were phenotype overlap among the various subtypes of PHP. Detection of GNAS gene methylation in patients with clinical suspicion of Type Ib PHP is helpful for the diagnosis and treatment of PHP.
Humans ; Chromogranins/genetics* ; GTP-Binding Protein alpha Subunits, Gs/genetics* ; Hypokalemia ; Calcium ; Pseudohypoparathyroidism/genetics* ; Phosphorus

OBJECTIVE: To preliminarily investigate the relationship between stimulatory G protein α subunit (GNAS) and thyroid hormone receptor α (THRA) gene mutations and clinical phenotypes in children with congenital hypothyroidism (CH). METHODS: A total of 70 children with CH diagnosed by neonatal screening were enrolled. Their peripheral blood samples were collected to extract genomic DNA. GNAS and THRA genes were screened for mutations using next-generation sequencing. Bioinformatics software was used to analyze the pathogenicity of gene mutations. RESULTS: Of the 70 children with CH, nine missense mutations (three known mutations and six novel mutations) in the GNAS gene were detected in three patients (4%), and one gene polymorphism, c.508A>G(p.I170V), in the THRA gene was detected in four patients. The analysis results of bioinformatics software and ACMG/AMP guidelines showed that the two GNAS gene mutations [c.301C>T(p.R101C) and c.334G>A(p.E112K)] were more likely to be pathogenic. Three children with GNAS gene mutations showed different degrees of hypothyroidism. CONCLUSIONS GNAS gene mutations are related to the development of CH, and children with CH have different clinical manifestations. THRA gene mutations may not be associated with CH.
Chromogranins ; genetics ; Congenital Hypothyroidism ; GTP-Binding Protein alpha Subunits, Gs ; genetics ; Genes, erbA ; Humans ; Infant, Newborn ; Mutation ; Phenotype ; Thyroid Hormone Receptors alpha ; genetics

Wnt signaling has emerged as a major regulator of tissue development by governing the self-renewal and maintenance of stem cells in most tissue types. As a key upstream regulator of the Wnt pathway, the transmembrane E3 ligase ZNRF3 has recently been established to play a role in negative regulation of Wnt signaling by targeting Frizzled (FZD) receptor for ubiquitination and degradation. However, the upstream regulation of ZNRF3, in particular the turnover of ZNRF3, is still unclear. Here we report that ZNRF3 is accumulated in the presence of proteasome inhibitor treatment independent of its E3-ubiquitin ligase activity. Furthermore, the Cullin 1-specific SCF complex containing β-TRCP has been identified to directly interact with and ubiquitinate ZNRF3 thereby regulating its protein stability. Similar with the degradation of β-catenin by β-TRCP, ZNRF3 is ubiquitinated by β-TRCP in both CKI-phosphorylation- and degron-dependent manners. Thus, our findings not only identify a novel substrate for β-TRCP oncogenic regulation, but also highlight the dual regulation of Wnt signaling by β-TRCP in a context-dependent manner where β-TRCP negatively regulates Wnt signaling by targeting β-catenin, and positively regulates Wnt signaling by targeting ZNRF3.
Cells, Cultured ; Humans ; Proteolysis ; Ubiquitin-Protein Ligases ; metabolism ; Ubiquitination ; beta-Transducin Repeat-Containing Proteins ; metabolism