RESUMO
BACKGROUND: Imprinted genes play important functions in placentation and pregnancy; however, research on their roles in different placental diseases is limited. It is believed that epigenetic alterations, such as DNA methylation, of placental imprinting genes may contribute to the different pathological features of severe placental diseases, such as pre-eclampsia (PE) and placenta accreta spectrum disorders (PAS). RESULTS: In this study, we conducted a comparative analysis of the methylation and expression of placental imprinted genes between PE and PAS using bisulfite sequencing polymerase chain reaction (PCR) and quantitative PCR, respectively. Additionally, we assessed oxidative damage of placental DNA by determining 8-hydroxy-2'-deoxyguanosine levels and fetal growth by determining insulin-like growth factor 2 (IGF2) and cortisol levels in the umbilical cord blood using enzyme-linked immunosorbent assay. Our results indicated that methylation and expression of potassium voltage-gated channel subfamily Q member 1, GNAS complex locus, mesoderm specific transcript, and IGF2 were significantly altered in both PE and PAS placentas. Additionally, our results revealed that the maternal imprinted genes were significantly over-expressed in PE and significantly under-expressed in PAS compared with a normal pregnancy. Moreover, DNA oxidative damage was elevated and positively correlated with IGF2 DNA methylation in both PE and PAS placentas, and cortisol and IGF2 levels were significantly decreased in PE and PAS. CONCLUSIONS: This study suggested that DNA methylation and expression of imprinted genes are aberrant in both PE and PAS placentas and that PE and PAS have different methylation profiles, which may be linked to their unique pathogenesis.
Assuntos
Metilação de DNA , Impressão Genômica , Fator de Crescimento Insulin-Like II , Pré-Eclâmpsia , Humanos , Feminino , Gravidez , Metilação de DNA/genética , Impressão Genômica/genética , Fator de Crescimento Insulin-Like II/genética , Pré-Eclâmpsia/genética , Adulto , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Placenta/metabolismo , Epigênese Genética/genética , Hidrocortisona/sangue , Doenças Placentárias/genética , Estresse Oxidativo/genética , Sangue Fetal/química , Sangue Fetal/metabolismo , Cromograninas , Proteínas , Canais de Potássio de Abertura Dependente da Tensão da MembranaRESUMO
This retrospective transcriptomic study leveraged bioinformatics and machine learning algorithms to identify novel gene biomarkers and explore immune cell infiltration profiles associated with chronic obstructive pulmonary disease (COPD). Utilizing an integrated analysis of metadata encompassing six gene expression omnibus (GEO) microarray datasets, 987 differentially expressed genes were identified. Further gene ontology and pathway enrichment analyses revealed the enrichment of these genes across various biological processes and pathways. Moreover, a systematic integration of two machine learning algorithms along with pathway-gene correlations identified six candidate biomarkers, which were validated in a separate cohort comprising six additional microarray datasets, ultimately identifying ADD3 and GNAS as diagnostic biomarkers for COPD. Subsequently, the diagnostic efficacy of ADD3 and GNAS was assessed, and the impact of their expression levels on overall survival was further evaluated and quantified in the validation cohort. Examination of immune cell subtype infiltration found increased proportions of cytotoxic CD8+ T cells, resting and activated NK cells, along with decreased M0 and M2 macrophages, in COPD versus control samples. Correlation analyses also uncovered significant associations between ADD3 and GNAS expression and infiltration of various immune cell types. In conclusion, this study elucidates crucial COPD diagnostic biomarkers and immune cell profiles which may illuminate the immunopathological drivers of COPD progression, representing personalized therapeutic targets warranting further investigation.
Assuntos
Biomarcadores , Biologia Computacional , Aprendizado de Máquina , Doença Pulmonar Obstrutiva Crônica , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Doença Pulmonar Obstrutiva Crônica/imunologia , Humanos , Biomarcadores/metabolismo , Biologia Computacional/métodos , Cromograninas/genética , Perfilação da Expressão Gênica , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Masculino , Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Feminino , Transcriptoma/genética , Idoso , Pessoa de Meia-Idade , Estudos Retrospectivos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismoRESUMO
BACKGROUND/AIMS: Osteosarcoma is a prevalent and aggressive primary malignant bone tumor affecting children and adolescents. Despite advancements in sequencing technologies, there remains a lack of reliable prognostic biomarkers and effective targeted therapies for osteosarcoma. This study focuses on identifying key prognostic genes, particularly the role of GNAS, in osteosarcoma progression. METHODS: Bioinformatics analyses were performed on osteosarcoma datasets from the Gene Expression Omnibus (GEO). Differential gene expression analysis, weighted correlation network analysis (WGCNA), and survival analysis identified potential prognostic hub genes. The expression and function of these genes were validated through immunohistochemistry and animal experiments. Specifically, the role of GNAS was investigated through siRNA-mediated knockdown in osteosarcoma cell lines and nude mice models. RESULTS: Five hub genes (PROP1, GNAS, CYP4F2, LHX3, CNGB1) were identified as significantly related to osteosarcoma prognosis. Among these, GNAS was found to be highly expressed in osteosarcoma tissues compared to normal tissues. Immunohistochemical analysis confirmed the elevated expression of GNAS in osteosarcoma samples. GNAS mutation analysis revealed a low mutation rate in osteosarcoma, suggesting its oncogenic role is independent of mutational status. Animal experiments demonstrated that knocking down GNAS significantly inhibited tumor growth and induced apoptosis in osteosarcoma cells. CONCLUSION: GNAS is highly expressed in osteosarcoma and associated with poor prognosis, acting as an oncogene in osteosarcoma progression. Targeting GNAS could be a potential therapeutic strategy for osteosarcoma. Further studies on GNAS-related signaling pathways may provide deeper insights into the molecular mechanisms driving osteosarcoma malignancy.
Assuntos
Neoplasias Ósseas , Cromograninas , Subunidades alfa Gs de Proteínas de Ligação ao GTP , Camundongos Nus , Osteossarcoma , Animais , Humanos , Camundongos , Apoptose , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Neoplasias Ósseas/metabolismo , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células , Cromograninas/genética , Cromograninas/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Camundongos Endogâmicos BALB C , Mutação , Osteossarcoma/genética , Osteossarcoma/patologia , Osteossarcoma/metabolismo , Prognóstico , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
A long-held tenet in inositol-lipid signaling is that cleavage of membrane phosphoinositides by phospholipase Cß (PLCß) isozymes to increase cytosolic Ca2+ in living cells is exclusive to Gq- and Gi-sensitive G protein-coupled receptors (GPCRs). Here we extend this central tenet and show that Gs-GPCRs also partake in inositol-lipid signaling and thereby increase cytosolic Ca2+. By combining CRISPR/Cas9 genome editing to delete Gαs, the adenylyl cyclase isoforms 3 and 6, or the PLCß1-4 isozymes, with pharmacological and genetic inhibition of Gq and G11, we pin down Gs-derived Gßγ as driver of a PLCß2/3-mediated cytosolic Ca2+ release module. This module does not require but crosstalks with Gαs-dependent cAMP, demands Gαq to release PLCß3 autoinhibition, but becomes Gq-independent with mutational disruption of the PLCß3 autoinhibited state. Our findings uncover the key steps of a previously unappreciated mechanism utilized by mammalian cells to finetune their calcium signaling regulation through Gs-GPCRs.
Assuntos
Sinalização do Cálcio , Cálcio , Fosfolipase C beta , Receptores Acoplados a Proteínas G , Humanos , Fosfolipase C beta/metabolismo , Fosfolipase C beta/genética , Células HEK293 , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Cálcio/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Sistemas CRISPR-Cas , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , AMP Cíclico/metabolismo , Animais , Edição de Genes , Citosol/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/genética , Adenilil Ciclases/metabolismo , Adenilil Ciclases/genéticaRESUMO
Despite selective HDAC3 inhibition showing promise in a subset of lymphomas with CREBBP mutations, wild-type tumors generally exhibit resistance. Here, using unbiased genome-wide CRISPR screening, we identify GNAS knockout (KO) as a sensitizer of resistant lymphoma cells to HDAC3 inhibition. Mechanistically, GNAS KO-induced sensitization is independent of the canonical G-protein activities but unexpectedly mediated by viral mimicry-related interferon (IFN) responses, characterized by TBK1 and IRF3 activation, double-stranded RNA formation, and transposable element (TE) expression. GNAS KO additionally synergizes with HDAC3 inhibition to enhance CD8+ T cell-induced cytotoxicity. Moreover, we observe in human lymphoma patients that low GNAS expression is associated with high baseline TE expression and upregulated IFN signaling and shares common disrupted biological activities with GNAS KO in histone modification, mRNA processing, and transcriptional regulation. Collectively, our findings establish an unprecedented link between HDAC3 inhibition and viral mimicry in lymphoma. We suggest low GNAS expression as a potential biomarker that reflects viral mimicry priming for enhanced response to HDAC3 inhibition in the clinical treatment of lymphoma, especially the CREBBP wild-type cases.
Assuntos
Cromograninas , Subunidades alfa Gs de Proteínas de Ligação ao GTP , Histona Desacetilases , Linfoma , Humanos , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Linfoma/genética , Linfoma/patologia , Linfoma/virologia , Linfoma/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Cromograninas/genética , Camundongos , Interferons/metabolismo , Animais , Inibidores de Histona Desacetilases/farmacologia , Linhagem Celular Tumoral , Técnicas de Inativação de Genes , Transdução de SinaisRESUMO
Diagnosis of biliopancreatic cancers by the available serum tumor markers, imaging, and histopathological tissue specimen examination remains a challenge. Circulating cell-free DNA derived from matched pairs of secretin-stimulated duodenal fluid (DF) and plasma from 10 patients with biliopancreatic diseases and 8 control subjects was analyzed using AmpliSeq™ HD technology for Ion Torrent Next-Generation Sequencing to evaluate the potential of liquid biopsy with DF in biliopancreatic cancers. The median cfDNA concentration was greater in DF-derived than in plasma-derived samples. A total of 13 variants were detected: 11 vs. 1 were exclusive for DF relative to the plasma source, and 1 was shared between the two body fluids. According to the four-tier systems, 10 clinical tier-I-II (76.9%), 1 tier-III (7.7%), and 2 tier-IV (15.4%) variants were identified. Notably, the 11 tier-I-III variants were exclusively found in DF-derived cfDNA from five patients with biliopancreatic cancers, and were detected in seven genes (KRAS, TP53, BRAF, CDKN2A, RNF43, GNAS, and PIK3CA); 82% of the tier-I-III variants had a low abundance, with a VAF < 6%. The mutational profiling of DF seems to be a reliable and promising tool for identifying cancer-associated alterations in malignant cancers of the biliopancreatic tract.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Neoplasias Pancreáticas , Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/patologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Duodeno/metabolismo , Duodeno/patologia , Biomarcadores Tumorais/genética , Biópsia Líquida/métodos , Adulto , Ácidos Nucleicos Livres/genética , Neoplasias do Sistema Biliar/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , CromograninasRESUMO
OBJECTIVES: Pseudohypoparathyroidism (PHP) comprises a cluster of heterogeneous diseases characterized by hypocalcemia and hyperphosphatemia due to parathyroid hormone (PTH) resistance. PHP type 1B (PHP1B) is caused by heterozygous maternal deletions within GNAS or STX16. STX16 exon 2-6 deletion is commonly observed in autosomal dominant (AD)-PHP1B, while sporadic PHP1B commonly results from methylation abnormalities of maternal differentially methylated regions and remains unclear at the molecular level. CASE PRESENTATION: A 39-year-old male patient with PHP1B, who had his first seizure at 15 years of age, presented to our hospital. The methylation-specific multiplex ligation-dependent probe amplification results showed a half-reduced copy number of STX16 exon 5-7 and loss of methylation at GNAS exon A/B. His mother also had a half-reduced copy number of STX16 exon 5-7 but with normal methylation of GNAS. His father has a normal copy number of STX16 and normal methylation of GNAS. CONCLUSIONS: For the recognition and early diagnosis of this kind of disease, here we report the clinical symptoms, auxiliary examinations, genetic testing characteristics, and treatment of the patient.
Assuntos
Éxons , Pseudo-Hipoparatireoidismo , Sintaxina 16 , Humanos , Masculino , Pseudo-Hipoparatireoidismo/genética , Pseudo-Hipoparatireoidismo/complicações , Adulto , Sintaxina 16/genética , Éxons/genética , Deleção de Sequência , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Prognóstico , Cromograninas/genéticaRESUMO
PURPOSE: To investigate whether the DNA methylation profiles of GNAS(20q13.32), MEST(7q32.2), MESTIT1(7q32.2), IGF2(11p15.5), H19 (7q32.2), and CEP41(7q32.2) genes are related to the transcriptomic and epigenomic etiology of male infertility. METHODS: The DNA methylation levels of spermatozoa were obtained from fertile (n = 30), oligozoospermic (n = 30), and men with normal sperm count (n = 30). The methylation status of each CpG site was categorized as hypermethylated or hypomethylated. Expression levels of target gene transcripts were determined using real-time PCR. RESULTS: The oligozoospermia showed a higher frequency of hypermethylation at GNASAS 1st, 3rd, and 5th CpG dinucleotides (66.7%, 73.3%, 73.3%) compared to the fertile group (33.3%, 33.3%, 40%, respectively). The normal sperm count exhibited a higher frequency of hypermethylation at the 3rd CpG of CEP41 (46.7%) than the fertile group (16.7%). Normal sperm count was predicted by CEP41 hypermethylation (OR = 1.750, 95%CI 1.038-2.950) and hypermethylation of both CEP41 and GNASAS (OR = 2.389, 95%CI 1.137-5.021). Oligozoospermia was predicted solely by GNASAS hypermethylation (OR = 2.460, 95%CI 1.315-4.603). In sperms with decreased IGF2 expression in the fertile group, we observed hypomethylation in the 2nd CpG of IGF2 antisense (IFG2AS), and hypermethylation in the 1st, 2nd, and 4th CpGs of H19. No significant relationship was found between IGF2 expression and methylation status of IGF2AS and H19 in infertile groups. CONCLUSION: The disappearance of the relationship between IGF2 expression and IGF2AS and H19 methylations in the infertile group provides new information regarding the disruption of epigenetic programming during spermatogenesis. A better understanding of sperm GNASAS and CEP41 hypermethylation could advance innovative diagnostic markers for male infertility.
Assuntos
Cromograninas , Metilação de DNA , Subunidades alfa Gs de Proteínas de Ligação ao GTP , Impressão Genômica , Infertilidade Masculina , Oligospermia , Masculino , Humanos , Metilação de DNA/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Cromograninas/genética , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Impressão Genômica/genética , Adulto , Oligospermia/genética , Oligospermia/patologia , Espermatozoides/patologia , Espermatozoides/metabolismo , Fator de Crescimento Insulin-Like II/genética , Epigênese Genética/genética , Ilhas de CpG/genética , RNA Longo não Codificante/genética , Contagem de EspermatozoidesRESUMO
The genetic era has opened the opportunity of using personalized therapeutic approaches, in part based on targeting genes with somatic mutations. For example, lymphomas harboring the highly recurrent CREBBP mutation show dependency on HDAC3, thus selective inhibition of HDAC3 reversed the epigenetic effects of CREBBP mutation, halted lymphoma growth, and induced MHC class II expression, enabling the T-cells to recognize and kill lymphoma cells. However, CREBBP wild type (WT) cells are less sensitive to this approach. In this issue of Leukemia, He et al. have executed a genome-wide CRISPR screening that identified GNAS as a target to maximize the therapeutic activity of HDAC3 inhibition in CREBBP WT lymphoma.
Assuntos
Proteína de Ligação a CREB , Cromograninas , Subunidades alfa Gs de Proteínas de Ligação ao GTP , Histona Desacetilases , Linfoma de Células B , Humanos , Proteína de Ligação a CREB/genética , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Cromograninas/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Linfoma de Células B/genética , Linfoma de Células B/patologia , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Inativação GênicaRESUMO
Approximately 30%-40% of growth hormone-secreting pituitary adenomas (GHPAs) harbor somatic activating mutations in GNAS (α subunit of stimulatory G protein). Mutations in GNAS are associated with clinical features of smaller and less invasive tumors. However, the role of GNAS mutations in the invasiveness of GHPAs is unclear. GNAS mutations were detected in GHPAs using a standard polymerase chain reaction (PCR) sequencing procedure. The expression of mutation-associated maternally expressed gene 3 (MEG3) was evaluated with RT-qPCR. MEG3 was manipulated in GH3 cells using a lentiviral expression system. Cell invasion ability was measured using a Transwell assay, and epithelial-mesenchymal transition (EMT)-associated proteins were quantified by immunofluorescence and western blotting. Finally, a tumor cell xenograft mouse model was used to verify the effect of MEG3 on tumor growth and invasiveness. The invasiveness of GHPAs was significantly decreased in mice with mutated GNAS compared with that in mice with wild-type GNAS. Consistently, the invasiveness of mutant GNAS-expressing GH3 cells decreased. MEG3 is uniquely expressed at high levels in GHPAs harboring mutated GNAS. Accordingly, MEG3 upregulation inhibited tumor cell invasion, and conversely, MEG3 downregulation increased tumor cell invasion. Mechanistically, GNAS mutations inhibit EMT in GHPAs. MEG3 in mutated GNAS cells prevented cell invasion through the inactivation of the Wnt/ß-catenin signaling pathway, which was further validated in vivo. Our data suggest that GNAS mutations may suppress cell invasion in GHPAs by regulating EMT through the activation of the MEG3/Wnt/ß-catenin signaling pathway.
Assuntos
Cromograninas , Subunidades alfa Gs de Proteínas de Ligação ao GTP , Adenoma Hipofisário Secretor de Hormônio do Crescimento , Mutação , Invasividade Neoplásica , RNA Longo não Codificante , Adulto , Animais , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Linhagem Celular Tumoral , Proliferação de Células/genética , Cromograninas/genética , Cromograninas/metabolismo , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Adenoma Hipofisário Secretor de Hormônio do Crescimento/genética , Adenoma Hipofisário Secretor de Hormônio do Crescimento/patologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Invasividade Neoplásica/genética , RNA Longo não Codificante/genética , Via de Sinalização Wnt/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Introduction: The dysregulation of cell fate toward osteoprecursor cells associated with most GNAS-based disorders may lead to episodic de novo extraskeletal or ectopic bone formation in subcutaneous tissues. The bony lesion distribution suggests the involvement of abnormal differentiation of mesenchymal stem cells (MSCs) and/or more committed precursor cells. Data from transgenic mice support the concept that GNAS is a crucial factor in regulating lineage switching between osteoblasts (OBs) and adipocyte fates. The mosaic nature of heterotopic bone lesions suggests that GNAS genetic defects provide a sensitized background for ectopic osteodifferentiation, but the underlying molecular mechanism remains largely unknown. Methods: The effect of GNAS silencing in the presence and/or absence of osteoblastic stimuli was evaluated in the human L88/5 MSC line during osteodifferentiation. A comparison of the data obtained with data coming from a bony lesion from a GNAS-mutated patient was also provided. Results: Our study adds some dowels to the current fragmented notions about the role of GNAS during osteoblastic differentiation, such as the premature transition of immature OBs into osteocytes and the characterization of the differences in the deposed bone matrix. Conclusion: We demonstrated that our cell model partially replicates the in vivo behavior results, resulting in an applicable human model to elucidate the pathophysiology of ectopic bone formation in GNAS-based disorders.
Assuntos
Diferenciação Celular , Cromograninas , Subunidades alfa Gs de Proteínas de Ligação ao GTP , Osteoblastos , Osteogênese , Humanos , Diferenciação Celular/genética , Linhagem Celular , Cromograninas/genética , Inativação Gênica , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Osteoblastos/metabolismo , Osteoblastos/citologia , Osteogênese/genéticaRESUMO
Chemoresistance is one of the major causes of poor prognosis in osteosarcoma. Alternative therapeutic strategies for osteosarcoma are limited, indicating that increasing sensitivity to currently used chemotherapies could be an effective approach to improve patient outcomes. Using a kinome-wide CRISPR screen, we identified PRKDC as a critical determinant of doxorubicin (DOX) sensitivity in osteosarcoma. The analysis of clinical samples demonstrated that PRKDC was hyperactivated in osteosarcoma, and functional experiments showed that the loss of PRKDC significantly increased sensitivity of osteosarcoma to DOX. Mechanistically, PRKDC recruited and bound GDE2 to enhance the stability of protein GNAS. The elevated GNAS protein levels subsequently activated AKT phosphorylation and conferred resistance to DOX. The PRKDC inhibitor AZD7648 and DOX synergized and strongly suppressed the growth of osteosarcoma in mouse xenograft models and human organoids. In conclusion, the PRKDC-GDE2-GNAS-AKT regulatory axis suppresses DOX sensitivity and comprises targetable candidates for improving the efficacy of chemotherapy in osteosarcoma. Significance: Targeting PRKDC suppresses AKT activation and increases sensitivity to doxorubicin in osteosarcoma, which provides a therapeutic strategy for overcoming chemoresistance.
Assuntos
Neoplasias Ósseas , Cromograninas , Doxorrubicina , Resistencia a Medicamentos Antineoplásicos , Subunidades alfa Gs de Proteínas de Ligação ao GTP , Osteossarcoma , Proteínas Proto-Oncogênicas c-akt , Ensaios Antitumorais Modelo de Xenoenxerto , Osteossarcoma/tratamento farmacológico , Osteossarcoma/patologia , Osteossarcoma/metabolismo , Osteossarcoma/genética , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Camundongos , Doxorrubicina/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/patologia , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/genética , Cromograninas/genética , Cromograninas/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Linhagem Celular Tumoral , Camundongos Nus , Antibióticos Antineoplásicos/farmacologia , Feminino , Proliferação de Células/efeitos dos fármacosRESUMO
Activation of G proteins through nucleotide exchange initiates intracellular signaling cascades essential for life processes. Under normal conditions, nucleotide exchange is regulated by the formation of G protein-G protein-coupled receptor complexes. Single point mutations in the Gα subunit of G proteins bypass this interaction, leading to loss of function or constitutive gain of function, which is closely linked with the onset of multiple diseases. Despite the recognized significance of Gα mutations in disease pathology, structural information for most variants is lacking, potentially due to inherent protein dynamics that pose challenges for crystallography. To address this, we leveraged an integrative spectroscopic and computational approach to structurally characterize seven of the most frequently observed and clinically relevant mutations in the stimulatory Gα subunit, GαS. A previously proposed allosteric model of Gα activation linked structural changes in the nucleotide-binding pocket with functionally important changes in interactions between switch regions. We investigated this allosteric connection in GαS by integrating data from variable temperature CD spectroscopy, which measured changes in global protein structure and stability, and molecular dynamics simulations, which observed changes in interaction networks between GαS switch regions. Additionally, saturation-transfer difference NMR spectroscopy was applied to observe changes in nucleotide interactions with residues within the nucleotide binding site. These data have enabled testing of predictions regarding how mutations in GαS result in loss or gain of function and evaluation of proposed structural mechanisms. The integration of experimental and computational data allowed us to propose a more nuanced classification of mechanisms underlying GαS gain-of-function and loss-of-function mutations.
Assuntos
Simulação de Dinâmica Molecular , Humanos , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Mutação , Regulação AlostéricaRESUMO
Keloids are pathological scar tissue resulting from skin trauma or spontaneous formation, often accompanied by itching and pain. Although GNAS antisense RNA 1 (GNAS-AS1) shows abnormal upregulation in keloids, the underlying molecular mechanism is unclear. The levels of genes and proteins in clinical tissues from patients with keloids and human keloid fibroblasts (HKFs) were measured using quantitative reverse transcription PCR, western blot and enzyme-linked immunosorbent assay. The features of HKFs, including proliferation and migration, were evaluated using cell counting kit 8 and a wound healing assay. The colocalization of GNAS-AS1 and miR-196a-5p in HKFs was measured using fluorescence in situ hybridization. The relationships among GNAS-AS1, miR-196a-5p and C-X-C motif chemokine ligand 12 (CXCL12) in samples from patients with keloids were analysed by Pearson correlation analysis. Gene interactions were validated by chromatin immunoprecipitation and luciferase reporter assays. GNAS-AS1 and CXCL12 expression were upregulated and miR-196a-5p expression was downregulated in clinical tissues from patients with keloids. GNAS-AS1 knockdown inhibited proliferation, migration, and extracellular matrix (ECM) accumulation of HKFs, all of which were reversed by miR-196a-5p downregulation. Signal transducer and activator of transcription 3 (STAT3) induced GNAS-AS1 transcription through GNAS-AS1 promoter interaction, and niclosamide, a STAT3 inhibitor, decreased GNAS-AS1 expression. GNAS-AS1 positively regulated CXCL12 by sponging miR-196-5p. Furthermore, CXCL12 knockdown restrained STAT3 phosphorylation in HKFs. Our findings revealed a feedback loop of STAT3/GNAS-AS1/miR-196a-5p/CXCL12/STAT3 that promoted HKF proliferation, migration and ECM accumulation and affected keloid progression.
Assuntos
Proliferação de Células , Quimiocina CXCL12 , Fibroblastos , Queloide , MicroRNAs , RNA Longo não Codificante , Fator de Transcrição STAT3 , Queloide/metabolismo , Queloide/genética , Queloide/patologia , Humanos , MicroRNAs/metabolismo , MicroRNAs/genética , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Quimiocina CXCL12/metabolismo , Quimiocina CXCL12/genética , Fibroblastos/metabolismo , Movimento Celular , Retroalimentação Fisiológica , Cromograninas/genética , Cromograninas/metabolismo , Masculino , Feminino , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Transdução de Sinais , Adulto , Células Cultivadas , Regulação para CimaRESUMO
The study of circulating tumor DNA (ctDNA) plays a pivotal role in advancing precision oncology, providing valuable information for individualized patient care and contributing to the ongoing effort to improve cancer diagnosis, treatment, and management. However, its applicability in pseudomyxoma peritonei (PMP) remains unexplored. In this multicenter retrospective study involving 21 PMP patients, we investigated ctDNA presence in peripheral blood using three distinct methodologies. Despite mucinous tumor tissues exhibiting KRAS and GNAS mutations, ctDNA for these mutations was undetectable in blood samples. In this pilot study, circulating tumor DNA was not detected in blood when the tumor harbored mutations of known significance. In the future, a study with a larger sample size is needed to confirm these findings and to determine whether ctDNA could identify patients at risk for early recurrence and/or systemic metastases.
Assuntos
DNA Tumoral Circulante , Neoplasias Peritoneais , Pseudomixoma Peritoneal , Humanos , Pseudomixoma Peritoneal/genética , Pseudomixoma Peritoneal/sangue , Pseudomixoma Peritoneal/patologia , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/secundário , Neoplasias Peritoneais/sangue , DNA Tumoral Circulante/genética , DNA Tumoral Circulante/sangue , Estudos Retrospectivos , Feminino , Pessoa de Meia-Idade , Masculino , Idoso , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Cromograninas/genética , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Projetos Piloto , AdultoRESUMO
PURPOSE: To summarize the clinical manifestations of craniofacial fibrous dysplasia (CFD) patients with ocular complications, and find effective methods to diagnose early. METHODS: Nine CFD patients with ocular complications, and their parents were recruited in this study. All patients underwent ocular and systemic examinations. Bone lesions from all patients and peripheral blood from patients and their parents were collected for whole exome sequencing (WES). According to the screening for low-frequency deleterious variants, and bioinformatics variants prediction software, possible disease-causing variants were found in multiple CFD patients. The variants were validated by Sanger sequencing. Trio analysis was performed to verify the genetic patterns of CFD. RESULTS: All patients were diagnosed with CFD, according to the clinical manifestations, classic radiographic appearance, and pathological biopsy. The main symptoms of the 9 CFD patients, included visual decline (9/9), craniofacial deformity (3/9) and strabismus (2/9), with few extraocular manifestations. The family backgrounds of all the CFD patients indicated that only the patient was affected, and their immediate family members were normal. GNAS variants were identified in all bone lesions from CFD patients, including two variant types: c.601C > T:p.R201C(6/9) and c.602G > A:p.R201H (3/9) in exon 8. The detection rate reached 100% by WES, but only 77.8% by Sanger sequencing. Interestingly, we found GNAS variants could not be detected in peripheral blood samples from CFD patients or their parents, and other potentially disease-causing gene variants related to CFD were not found. CONCLUSIONS: For CFD patients with bone lesions involving the optic canal or sphenoid sinus regions, ocular symptoms should also be considered. Furthermore, we confirmed that CFD is not inherited, somatic variants in the GNAS gene are the main pathogenic gene causing CFD. Compared to the traditional methods in molecular genetic diagnosis of CFD, WES is more feasible and effective but limited in the type of samples.
Assuntos
Displasia Fibrosa Craniofacial , Sequenciamento do Exoma , Humanos , Masculino , Feminino , Criança , Adolescente , Displasia Fibrosa Craniofacial/genética , Displasia Fibrosa Craniofacial/diagnóstico , Adulto , Adulto Jovem , Mutação , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Cromograninas/genética , Análise Mutacional de DNA , Pré-Escolar , Linhagem , Técnicas de Diagnóstico Molecular/métodos , Estrabismo/genética , Estrabismo/diagnósticoRESUMO
Cellular myxoma is a benign soft tissue tumor frequently associated with GNAS mutation that may morphologically resemble low-grade myxofibrosarcoma. This study aimed to identify the undescribed methylation profile of cellular myxoma and compare it to myxofibrosarcoma. We performed molecular analysis on twenty cellular myxomas and nine myxofibrosarcomas and analyzed the results using the methylation-based DKFZ sarcoma classifier. A total of 90% of the cellular myxomas had GNAS mutations (four loci had not been previously described). Copy number variations were found in all myxofibrosarcomas but in none of the cellular myxomas. In the classifier, none of the cellular myxomas reached the 0.9 threshold. Unsupervised t-SNE analysis demonstrated that cellular myxomas form their own clusters, distinct from myxofibrosarcomas. Our study shows the diagnostic potential and the limitations of molecular analysis in cases where morphology and immunohistochemistry are not sufficient to distinguish cellular myxoma from myxofibrosarcoma, particularly regarding GNAS wild-type tumors. The DKFZ sarcoma classifier only provided a valid prediction for one myxofibrosarcoma case; this limitation could be improved by training the tool with a more considerable number of cases. Additionally, the classifier should be introduced to a broader spectrum of mesenchymal neoplasms, including benign tumors like cellular myxoma, whose distinct methylation pattern we demonstrated.
Assuntos
Variações do Número de Cópias de DNA , Metilação de DNA , Fibrossarcoma , Mixoma , Humanos , Mixoma/genética , Mixoma/diagnóstico , Mixoma/patologia , Fibrossarcoma/genética , Fibrossarcoma/patologia , Fibrossarcoma/diagnóstico , Fibrossarcoma/metabolismo , Pessoa de Meia-Idade , Feminino , Idoso , Masculino , Adulto , Mutação , Diagnóstico Diferencial , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Cromograninas/genética , Idoso de 80 Anos ou mais , Neoplasias de Tecidos Moles/genética , Neoplasias de Tecidos Moles/diagnóstico , Neoplasias de Tecidos Moles/patologiaRESUMO
Fibrous dysplasia (FD) is a mosaic skeletal disorder caused by somatic activating variants of GNAS encoding for Gαs and leading to excessive cyclic adenosine monophosphate signaling in bone-marrow stromal cells (BMSCs). The effect of Gαs activation in the BMSC transcriptome and how it influences FD lesion microenvironment are unclear. We analyzed changes induced by Gαs activation in the BMSC transcriptome and secretome. RNAseq analysis of differential gene expression of cultured BMSCs from patients with FD and healthy volunteers, and from an inducible mouse model of FD, was performed, and the transcriptomic profiles of both models were combined to build a robust FD BMSC genetic signature. Pathways related to Gαs activation, cytokine signaling, and extracellular matrix deposition were identified. To assess the modulation of several key secreted factors in FD pathogenesis, cytokines and other factors were measured in culture media. Cytokines were also screened in a collection of plasma samples from patients with FD, and positive correlations of several cytokines to their disease burden score, as well as to one another and bone turnover markers, were found. These data support the pro-inflammatory, pro-osteoclastic behavior of FD BMSCs and point to several cytokines and other secreted factors as possible therapeutic targets and/or circulating biomarkers for FD.
Assuntos
Displasia Fibrosa Óssea , Células-Tronco Mesenquimais , Transcriptoma , Humanos , Animais , Células-Tronco Mesenquimais/metabolismo , Transcriptoma/genética , Camundongos , Displasia Fibrosa Óssea/genética , Displasia Fibrosa Óssea/metabolismo , Displasia Fibrosa Óssea/patologia , Masculino , Feminino , Citocinas/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Adulto , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Pseudohypoparathyroidism (PHP) is caused by loss-of-function mutations at the GNAS gene (as in the PHP type 1A; PHP1A), de novo or inherited at heterozygous state, or by epigenetic alterations at the GNAS locus (as in the PHP1B). The condition of PHP refers to a heterogeneous group of disorders that share common clinical and biological features of PTH resistance. Manifestations related to resistance to other hormones are also reported in many patients with PHP, in association with the phenotypic picture of Albright hereditary osteodystrophy characterized by short stature, round facies, subcutaneous ossifications, brachydactyly, mental retardation and, in some subtypes, obesity. The purpose of our study is to report a new mutation in the GNAS gene and to describe the significant phenotypic variability of three sisters with PHP1A bearing the same mutation. CASE PRESENTATION: We describe the cases of three sisters with PHP1A bearing the same mutation but characterized by a significantly different phenotypic picture at onset and during follow-up in terms of clinical features, auxological pattern and biochemical changes. Clinical exome sequencing revealed a never before described heterozygote mutation in the GNAS gene (NM_000516.5 c.118_139 + 51del) of autosomal dominant maternal transmission in the three siblings, confirming the diagnosis of PHP1A. CONCLUSIONS: This study reported on a novel mutation of GNAS gene and highlighted the clinical heterogeneity of PHP1A characterized by wide genotype-phenotype variability. The appropriate diagnosis has crucial implications for patient care and long-term multidisciplinary follow-up.
Assuntos
Cromograninas , Subunidades alfa Gs de Proteínas de Ligação ao GTP , Pseudo-Hipoparatireoidismo , Humanos , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Pseudo-Hipoparatireoidismo/genética , Pseudo-Hipoparatireoidismo/diagnóstico , Cromograninas/genética , Feminino , Criança , Fenótipo , Linhagem , Mutação , Adolescente , Pré-EscolarRESUMO
OBJECTIVES: Inactivating GNAS mutations result in varied phenotypes depending on parental origin. Maternally inherited mutations typically lead to hormone resistance and Albright's hereditary osteodystrophy (AHO), characterised by short stature, round facies, brachydactyly and subcutaneous ossifications. Paternal inheritance presents with features of AHO or ectopic ossification without hormone resistance. This report describes the case of a child with osteoma cutis and medulloblastoma. The objective of this report is to highlight the emerging association between inactivating germline GNAS mutations and medulloblastoma, aiming to shed light on its implications for tumor biology and promote future development of targeted surveillance strategies to improve outcomes in paediatric patients with these mutations. CASE PRESENTATION: A 12-month-old boy presented with multiple plaque-like skin lesions. Biopsy confirmed osteoma cutis, prompting genetic testing which confirmed a heterozygous inactivating GNAS mutation. At 2.5 years of age, he developed neurological symptoms and was diagnosed with a desmoplastic nodular medulloblastoma, SHH molecular group, confirmed by MRI and histology. Further analysis indicated a biallelic loss of GNAS in the tumor. CONCLUSIONS: This case provides important insights into the role of GNAS as a tumor suppressor and the emerging association between inactivating GNAS variants and the development of medulloblastoma. The case underscores the importance of careful neurological assessment and ongoing vigilance in children with known inactivating GNAS variants or associated phenotypes. Further work to establish genotype-phenotype correlations is needed to inform optimal management of these patients.