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*

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

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

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 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

BACKGROUNDThe addition of anti-human epidermal growth factor receptor 2 (HER2)-targeted drugs, such as trastuzumab, lapatinib, and trastuzumab emtansine (T-DM1), to chemotherapy significantly improved prognosis of HER2-positive breast cancer patients. However, it was confused that metastatic patients vary in the response of targeted drug. Therefore, methods of accurately predicting drug response were really needed. To overcome the spatial and temporal limitations of biopsies, we aimed to develop a more sensitive and less invasive method of detecting mutations associated with anti-HER2 therapeutic response through circulating-free DNA (cfDNA).

METHODSFrom March 6, 2014 to December 10, 2014, 24 plasma samples from 20 patients with HER2-positive metastatic breast cancer who received systemic therapy were eligible. We used a panel for detection of hot-spot mutations from 50 oncogenes and tumor suppressor genes, and then used targeted next-generation sequencing (NGS) to identify somatic mutation of these samples in those 50 genes. Samples taken before their first trastuzumab administration and subsequently proven with clinical benefit were grouped into sensitive group. The others were collected after disease progression of the trastuzumab-based therapy and were grouped into the resistant group.

RESULTSA total of 486 single-nucleotide variants from 46 genes were detected. Of these 46 genes, phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), proto-oncogene c-Kit (KIT), and tumor protein p53 (TP53) were the most common mutated genes. Seven genes, including epidermal growth factor receptor (EGFR), G protein subunit alpha S (GNAS), HRas proto-oncogene (HRAS), mutL homolog 1 (MLH1), cadherin 1 (CDH1), neuroblastoma RAS viral oncogene homolog (NRAS), and NOTCH1, that only occurred m utations in the resistant group were associated with the resistance of targeted therapy. In addition, we detected a HER2 S855I mutation in two patients who had persistent benefits from anti-HER2 therapy.

CONCLUSIONTargeted NGS of cfDNA has potential clinical utility to detect biomarkers from HER2-targeted therapies.

Adolescent ; Adult ; Aged ; Biomarkers, Tumor ; genetics ; Breast Neoplasms ; genetics ; metabolism ; Cadherins ; genetics ; Chromogranins ; genetics ; Class I Phosphatidylinositol 3-Kinases ; Drug Resistance, Neoplasm ; genetics ; Female ; GTP-Binding Protein alpha Subunits, Gs ; genetics ; Humans ; Male ; Middle Aged ; Mutation ; genetics ; Phosphatidylinositol 3-Kinases ; genetics ; Proto-Oncogene Proteins c-kit ; genetics ; Receptor, ErbB-2 ; metabolism ; Receptor, Notch1 ; genetics ; Tumor Suppressor Protein p53 ; genetics ; Young Adult

PURPOSE: Apoptosis of vascular endothelial cells is a type of endothelial damage that is associated with the pathogenesis of cardiovascular diseases such as atherosclerosis. Heterotrimeric GTP-binding proteins (G proteins), including the alpha 12 subunit of G protein (Galpha12), have been found to modulate cellular proliferation, differentiation, and apoptosis of numerous cell types. However, the role of Galpha12 in the regulation of apoptosis of vascular cells has not been elucidated. We investigated the role of Galpha12 in serum withdrawal-induced apoptosis of human umbilical vein endothelial cells (HUVECs) and its underlying mechanisms. MATERIALS AND METHODS: HUVECs were transfected with Galpha12 small-interfering RNA (siRNA) to knockdown the endogenous Galpha12 expression and were serum-deprived for 6 h to induce apoptosis. The apoptosis of HUVECs were assessed by Western blotting and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The expressions of microRNAs were analyzed by quantitative real-time PCR. RESULTS: Knockdown of Galpha12 with siRNA augmented the serum withdrawal-induced apoptosis of HUVECs and markedly repressed the expression of microRNA-155 (miR-155). Serum withdrawal-induced apoptosis of HUVECs was inhibited by the overexpression of miR-155 and increased significantly due to the inhibition of miR-155. Notably, the elevation of miR-155 expression prevented increased apoptosis of Galpha12-deficient HUVECs. CONCLUSION: From these results, we conclude that Galpha12 protects HUVECs from serum withdrawal-induced apoptosis by retaining miR-155 expression. This suggests that Galpha12 might play a protective role in vascular endothelial cells by regulating the expression of microRNAs.
*Apoptosis ; Atherosclerosis/*blood/genetics/immunology ; Cell Proliferation ; Endothelial Cells/*metabolism ; GTP-Binding Protein alpha Subunits, G12-G13/*genetics ; Gene Expression Profiling ; Gene Expression Regulation ; Human Umbilical Vein Endothelial Cells/cytology ; Humans ; MicroRNAs/*metabolism ; Protective Agents ; *RNA, Small Interfering ; Real-Time Polymerase Chain Reaction ; *Transfection

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

The worldwide prevalence of obesity is steadily increasing, nearly doubling between 1980 and 2008. Obesity is often associated with insulin resistance, a major risk factor for type 2 diabetes mellitus (T2DM): a costly chronic disease and serious public health problem. The underlying cause of T2DM is a failure of the beta cells of the pancreas to continue to produce enough insulin to counteract insulin resistance. Most current T2DM therapeutics do not prevent continued loss of insulin secretion capacity, and those that do have the potential to preserve beta cell mass and function are not effective in all patients. Therefore, developing new methods for preventing and treating obesity and T2DM is very timely and of great significance. There is now considerable literature demonstrating a link between inhibitory guanine nucleotide-binding protein (G protein) and G protein-coupled receptor (GPCR) signaling in insulin-responsive tissues and the pathogenesis of obesity and T2DM. These studies are suggesting new and emerging therapeutic targets for these conditions. In this review, we will discuss inhibitory G proteins and GPCRs that have primary actions in the beta cell and other peripheral sites as therapeutic targets for obesity and T2DM, improving satiety, insulin resistance and/or beta cell biology.
Animals ; Diabetes Mellitus, Type 2/drug therapy/*metabolism ; GTP-Binding Protein alpha Subunits/genetics/*metabolism ; Humans ; Insulin-Secreting Cells/metabolism ; Obesity/drug therapy/*metabolism ; Receptor, Melatonin, MT2/genetics/*metabolism ; Receptors, Adrenergic, alpha-1/genetics/*metabolism ; Receptors, Prostaglandin/genetics/*metabolism

OBJECTIVETo analyze the association between T393C single nucleotide polymorphism (SNP) of GNAS1 gene and non-valvular atrial fibrillation (AF) in Chinese Han patients.

METHODSNinety patients with non-valvular AF and 90 healthy subjects were examined for T393C SNP of GNAS1 gene using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The allele genotypes and the distribution of allele frequencies were analyzed and compared between the two groups. The relationship between allele frequency distribution characteristics and the heart rate variability (HRV) were also studied for analysis of the association between T393C SNP of GNAS1 gene and the autonomic nervous activation in non-valvular AF.

RESULTSThe two groups showed a significant difference in the frequencies of genotypes of T393C SNP of GNAS1 gene and allele frequencies (P<0.01). CC genotype and T393C allele frequency were significantly increased in the case group. pNN50, LF, or LF/HF showed no significant difference between different genotypes (P<0.05).

CONCLUTIONSThe T393C SNP of GNAS1 gene is closely associated with non-valvular AF in Chinese Han patients.

Adult ; Aged ; Aged, 80 and over ; Alleles ; Asian Continental Ancestry Group ; genetics ; Atrial Fibrillation ; genetics ; metabolism ; physiopathology ; Chromogranins ; Female ; GTP-Binding Protein alpha Subunits, Gs ; genetics ; Gene Frequency ; Genotype ; Heart Rate ; Humans ; Male ; Middle Aged ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single Nucleotide ; Risk Factors