1.Efficient fusion expression of G13 domain derived from granulysin in Escherichia coli.
Xiaoqiang LIU ; Xiangdong ZHA ; Yazhong XIAO ; Jinhuan YANG ; Nengshu LI
Chinese Journal of Biotechnology 2009;25(2):235-241
The G13 domain derived from granulysin shows high antimicrobial activities against Gram-positive and Gram-negative bacteria but does not lyse Jurkat cells or liposomes. To explore a new approach for high expression of the G13 domain, we fused the sequence encoding G13 to thioredoxin (Trx) gene to construct the recombinant expression vector (pThioHisA-G13). A cyanogen bromide (CNBr) cleavage site was introduced between the Trx and G13 to facilitate final release of the recombinant G13. The recombinant expression vector, pThioHisA-G13, was transformed into E. coli BL21 (DE3). Upon induction by IPTG Trx-G13 fusion protein was expressed and took the form of inclusion bodies counting 58% (W/W) of total cellular proteins. The inclusion body was solved by urea (8 mol/L) and then cleaved by CNBr. We purified the recombinant peptide G13 by one-step cation exchange chromatography. Results of agarose diffuse assay analysis indicated that the recombinant G13 exhibited antibacterial activity. The procedure described in this study will provide a reliable and simple method for highly efficient production of some cationic antimicrobial peptides.
Anti-Infective Agents
;
metabolism
;
Antigens, Differentiation, T-Lymphocyte
;
genetics
;
Cyanogen Bromide
;
pharmacology
;
Escherichia coli
;
genetics
;
metabolism
;
GTP-Binding Protein alpha Subunits, G12-G13
;
biosynthesis
;
genetics
;
Inclusion Bodies
;
metabolism
;
Protein Structure, Tertiary
;
genetics
;
Recombinant Fusion Proteins
;
biosynthesis
;
genetics
;
Thioredoxins
;
genetics
;
Transfection
2.Genetic analysis of a child with early onset neurodevelopmental disorder with involuntary movement and a literature review.
Wenjing HU ; Hongjun FANG ; Jingwen TANG ; Zhen ZHOU ; Liwen WU
Chinese Journal of Medical Genetics 2023;40(4):385-389
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
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Computational Biology
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Genetic Counseling
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Genomics
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Mutation
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Neurodevelopmental Disorders/genetics*
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Dyskinesias
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GTP-Binding Protein alpha Subunits, Gi-Go
3.Gαi1/3 mediation of Akt-mTOR activation is important for RSPO3-induced angiogenesis.
Gang XU ; Li-Na QI ; Mei-Qing ZHANG ; Xue-Yun LI ; Jin-Long CHAI ; Zhi-Qing ZHANG ; Xia CHEN ; Qian WANG ; Ke-Ran LI ; Cong CAO
Protein & Cell 2023;14(3):217-222
4.Clinical and genetic characteristics for 4 patients with Type Ib pseudohypoparathyroidism.
Yujun WANG ; Wenjun YANG ; Ping JIN ; Liling ZHAO ; Honghui HE
Journal of Central South University(Medical Sciences) 2022;47(10):1461-1466
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
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Chromogranins/genetics*
;
GTP-Binding Protein alpha Subunits, Gs/genetics*
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Hypokalemia
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Calcium
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Pseudohypoparathyroidism/genetics*
;
Phosphorus
5.Galpha12 Protects Vascular Endothelial Cells from Serum Withdrawal-Induced Apoptosis through Regulation of miR-155.
Hyeon Jeong LEE ; Eun Jig LEE ; Miran SEO
Yonsei Medical Journal 2016;57(1):247-253
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
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Atherosclerosis/*blood/genetics/immunology
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Cell Proliferation
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Endothelial Cells/*metabolism
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GTP-Binding Protein alpha Subunits, G12-G13/*genetics
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Gene Expression Profiling
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Gene Expression Regulation
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Human Umbilical Vein Endothelial Cells/cytology
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Humans
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MicroRNAs/*metabolism
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Protective Agents
;
*RNA, Small Interfering
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Real-Time Polymerase Chain Reaction
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*Transfection
6.Cooperation of Gbeta and Galphaq Protein in Contractile Response of Cat Lower Esophageal Sphincter (LES).
The Korean Journal of Physiology and Pharmacology 2003;7(6):349-355
We previously shown that LES contraction depends on M3 receptors linked to PTX insensitive Gq protein and activation of PLC. This results in production of IP3, which mediates calcium release, and contraction through a CaM dependent pathway. In the esophagus ACh activates M2 receptors linked to PTX sensitive Gi3 protein, resulting in activation of PLD, presumably, production of DAG. We investigated the role of PLC isozymes which can be activated by Gq or Gbeta protein on ACh-induced contraction in LES and esophagus. Immunoblot analysis showed the presence of 3 types of PLC isozymes, PLC-beta1, PLC-beta3, and PLC-gamma1, but not PLC-beta2, PLC-beta4, PLC-gamma2, PLC-delta1, and PLC-delta2 from both LES and esophageal muscle. ACh produced contraction in a dose dependent manner in LES and esophageal muscle cells obtained by enzymatic digestion with collagenase. PLC-beta1 or PLC-beta3 antibody incubation reduced contraction in response to ACh in LES but not in esophageal permeabilized cells, but PLC-gamma1 antibody incubation did not have an inhibitory effect. The inhibition by PLC-beta1 or PLC-beta3 antibody on Ach-induced contraction was antibody concentration dependent. The combination with PLC-beta1 and PLC-beta3 antibody completely abolished the contraction, suggesting that PLC-beta1 and PLC-beta3 have a synergism to inhibit the contraction in LES. PLC-beta1, -beta3 or -gamma1 antibody did not reduce the contraction of LES cells in response to DAG (10 (-6) M), suggesting that this isozyme of PLC may not activate PKC. When Gq/11 antibody was incubated, the inhibitory effect of the incubation of PLC beta3, but not of PLC beta1 was additive (Fig. 6). In contrast, when Gbeta antibody was incubated, the inhibitory effect of the incubation of PLC beta1, but not of PLC beta3 was additive. This data suggest that Gq/11 or Gbeta may activate cooperatively different PLC isozyme, PLCbeta1 or PLCbeta3 respectively.
Animals
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Calcium
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Cats*
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Collagenases
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Digestion
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Esophageal Sphincter, Lower*
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Esophagus
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GTP-Binding Protein alpha Subunits, Gq-G11*
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Isoenzymes
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Muscle Cells
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Phospholipase C beta
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Type C Phospholipases
7.Palmitoylation of GNAQ/11 is critical for tumor cell proliferation and survival in GNAQ/11-mutant uveal melanoma.
Yan ZHANG ; Baoyuan ZHANG ; Yongyun LI ; Yuting DAI ; Jiaoyang LI ; Donghe LI ; Zhizhou XIA ; Jianming ZHANG ; Ping LIU ; Ming CHEN ; Bo JIAO ; Ruibao REN
Frontiers of Medicine 2022;16(5):784-798
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 (Gαq/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/11Q209L-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 Gαq/11 signaling for cell survival and proliferation than other melanoma cells without GNAQ/11 mutations. Furthermore, the palmitoylation inhibitor, 2-bromopalmitate, also specifically disrupted Gαq/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
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Mice
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Animals
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Lipoylation
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NIH 3T3 Cells
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Uveal Neoplasms/genetics*
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Melanoma/genetics*
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Cell Proliferation
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Proto-Oncogene Proteins c-bcl-2
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GTP-Binding Protein alpha Subunits, Gq-G11/genetics*
8.Simultaneous deletion of floxed genes mediated by CaMKIIalpha-Cre in the brain and in male germ cells: application to conditional and conventional disruption of Goalpha.
Chan Il CHOI ; Sang Phil YOON ; Jung Mi CHOI ; Sung Soo KIM ; Young Don LEE ; Lutz BIRNBAUMER ; Haeyoung SUH-KIM
Experimental & Molecular Medicine 2014;46(5):e93-
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
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Brain/*metabolism
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Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics
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Female
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GTP-Binding Protein alpha Subunits, Gi-Go/*genetics
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*Gene Deletion
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Gene Knockout Techniques/*methods
;
Male
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Mice
;
RNA, Untranslated/genetics
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Recombination, Genetic
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Spermatozoa/*metabolism
9.Bioinformatic analysis of differentially expressed proteins in the dorsal raphe nucleus of rats after continuous treatment with olanzapine.
Ping HUANG ; Jian Cheng ZHU ; Hua LI ; Yan Zhao WANG ; Yi Min TANG ; Qiang LIU
Journal of Southern Medical University 2022;42(8):1221-1229
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
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Chromatography, Liquid
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Computational Biology
;
Dorsal Raphe Nucleus
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Female
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GTP-Binding Protein alpha Subunits, Gi-Go
;
Male
;
Mice
;
Olanzapine/adverse effects*
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Proteomics
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Rats
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Rats, Sprague-Dawley
;
Tandem Mass Spectrometry
10.An analysis of GNAS and THRA gene mutations in children with congenital hypothyroidism.
Xiao-Yu CHEN ; Yong LIU ; Jian-Hua LIU ; Xiao-Song QIN
Chinese Journal of Contemporary Pediatrics 2019;21(7):680-684
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
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genetics
;
Congenital Hypothyroidism
;
GTP-Binding Protein alpha Subunits, Gs
;
genetics
;
Genes, erbA
;
Humans
;
Infant, Newborn
;
Mutation
;
Phenotype
;
Thyroid Hormone Receptors alpha
;
genetics