RESUMO
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
Glucagon, a hormone released from pancreatic α-cells, is critical for maintaining euglycemia and plays a key role in the pathophysiology of diabetes. To stimulate the development of new classes of therapeutic agents targeting glucagon release, key α-cell signaling pathways that regulate glucagon secretion need to be identified. Here, we focused on the potential importance of α-cell Gs signaling on modulating α-cell function. Studies with α-cell-specific mouse models showed that activation of α-cell Gs signaling causes a marked increase in glucagon secretion. We also found that intra-islet adenosine plays an unexpected autocrine/paracrine role in promoting glucagon release via activation of α-cell Gs-coupled A2A adenosine receptors. Studies with α-cell-specific Gαs knockout mice showed that α-cell Gs also plays an essential role in stimulating the activity of the Gcg gene, thus ensuring proper islet glucagon content. Our data suggest that α-cell enriched Gs-coupled receptors represent potential targets for modulating α-cell function for therapeutic purposes.
Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP , Células Secretoras de Glucagon , Glucagon , Camundongos Knockout , Transdução de Sinais , Glucagon/metabolismo , Animais , Células Secretoras de Glucagon/metabolismo , Camundongos , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Adenosina/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptor A2A de Adenosina/genética , Masculino , Camundongos Endogâmicos C57BL , Ilhotas Pancreáticas/metabolismoRESUMO
Formation and retrieval of remote contextual memory depends on cortical engram neurons that are defined during learning. Manipulation of astrocytic Gq and Gi associated G-protein coupled receptor (GPCR) signaling has been shown to affect memory processing, but little is known about the role of cortical astrocytic Gs-GPCR signaling in remote memory acquisition and the functioning of cortical engram neurons. We assessed this by chemogenetic manipulation of astrocytes in the medial prefrontal cortex (mPFC) of male mice, during either encoding or consolidation of a contextual fear memory, while simultaneously labeling cortical engram neurons. We found that stimulation of astrocytic Gs signaling during memory encoding and consolidation did not alter remote memory expression. In line with this, the size of the mPFC engram population and the recall-induced reactivation of these neurons was unaffected. Hence, our data indicate that activation of Gs-GPCR signaling in cortical astrocytes is not sufficient to alter memory performance and functioning of cortical engram neurons.
Assuntos
Astrócitos , Medo , Neurônios , Córtex Pré-Frontal , Transdução de Sinais , Animais , Astrócitos/metabolismo , Masculino , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/fisiologia , Transdução de Sinais/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Medo/fisiologia , Camundongos Endogâmicos C57BL , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Camundongos , Memória/fisiologia , Memória de Longo Prazo/fisiologiaRESUMO
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
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
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
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
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
The human calcium-sensing receptor (CaSR) detects fluctuations in the extracellular Ca2+ concentration and maintains Ca2+ homeostasis1,2. It also mediates diverse cellular processes not associated with Ca2+ balance3-5. The functional pleiotropy of CaSR arises in part from its ability to signal through several G-protein subtypes6. We determined structures of CaSR in complex with G proteins from three different subfamilies: Gq, Gi and Gs. We found that the homodimeric CaSR of each complex couples to a single G protein through a common mode. This involves the C-terminal helix of each Gα subunit binding to a shallow pocket that is formed in one CaSR subunit by all three intracellular loops (ICL1-ICL3), an extended transmembrane helix 3 and an ordered C-terminal region. G-protein binding expands the transmembrane dimer interface, which is further stabilized by phospholipid. The restraint imposed by the receptor dimer, in combination with ICL2, enables G-protein activation by facilitating conformational transition of Gα. We identified a single Gα residue that determines Gq and Gs versus Gi selectivity. The length and flexibility of ICL2 allows CaSR to bind all three Gα subtypes, thereby conferring capacity for promiscuous G-protein coupling.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP , Receptores de Detecção de Cálcio , Humanos , Cálcio/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Modelos Moleculares , Ligação Proteica , Multimerização Proteica , Receptores de Detecção de Cálcio/metabolismo , Receptores de Detecção de Cálcio/química , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Sítios de Ligação , Estrutura Secundária de Proteína , Especificidade por SubstratoRESUMO
PURPOSE: Cardiac myxomas (CMs) are the second most common benign primary cardiac tumors, mainly originating within the left atrium. Approximately 5% of CM cases are associated with Carney Complex (CNC), an autosomal dominant multiple neoplasia syndrome often caused by germline mutations in the protein kinase A regulatory subunit 1A (PRKAR1A). Data concerning PRKAR1A alterations in sporadic myxomas are variable and sparse, with PRKAR1A mutations reported to range from 0% to 87%. Therefore, we investigated the frequency of PRKAR1A mutations in sporadic CM using next-generation sequencing (NGS). Additionally, we explored mutations in the catalytic domain of the Protein Kinase A complex (PRKACA) and examined the presence of GNAS mutations as another potential driver. METHODS AND RESULTS: This study retrospectively collected histological and clinical data from 27 patients with CM. First, we ruled out the possibility of underlying CNC through clinical evaluations and standardized interviews for each patient. Second, we performed PRKAR1A immunohistochemistry (IHC) analysis and graded the reactivity of myxoma cells semi-quantitatively. NGS was then applied to analyze the coding regions of PRKAR1A, PRKACA, and GNAS in all 27 cases. Of the 27 sporadic CM cases, 13 (48%) harbored mutations in PRKAR1A. Among these 13 mutant cases, six displayed more than one mutation in PRKAR1A. Most of the identified mutations resulted in premature stop codons or affected splicing. In PRKAR1A mutant CM cases, the loss of PRKAR1A protein expression was significantly more common. In two cases with missense mutations, protein expression remained preserved. Furthermore, a single mutation was detected in the catalytic domain of the protein kinase A complex, while no GNAS mutations were found. CONCLUSION: We identified a relatively high frequency of PRKAR1A mutations in sporadic CM. These PRKAR1A mutations may also represent an important oncogenic mechanism in sporadic myxomas, as already known in CM cases associated with CNC.
Assuntos
Cromograninas , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico , Subunidades alfa Gs de Proteínas de Ligação ao GTP , Neoplasias Cardíacas , Mixoma , Humanos , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Cromograninas/genética , Neoplasias Cardíacas/genética , Neoplasias Cardíacas/patologia , Neoplasias Cardíacas/enzimologia , Pessoa de Meia-Idade , Feminino , Masculino , Mixoma/genética , Mixoma/patologia , Mixoma/enzimologia , Adulto , Idoso , Estudos Retrospectivos , Análise Mutacional de DNA , Predisposição Genética para Doença , Mutação , Adulto Jovem , Fenótipo , Sequenciamento de Nucleotídeos em Larga Escala , Adolescente , Complexo de Carney/genética , Complexo de Carney/enzimologia , Complexo de Carney/patologia , Biomarcadores Tumorais/genética , Subunidades Catalíticas da Proteína Quinase Dependente de AMP CíclicoRESUMO
G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating guanine nucleotide exchange in the Gα subunit1. To visualize this mechanism, we developed a time-resolved cryo-EM approach that examines the progression of ensembles of pre-steady-state intermediates of a GPCR-G-protein complex. By monitoring the transitions of the stimulatory Gs protein in complex with the ß2-adrenergic receptor at short sequential time points after GTP addition, we identified the conformational trajectory underlying G-protein activation and functional dissociation from the receptor. Twenty structures generated from sequential overlapping particle subsets along this trajectory, compared to control structures, provide a high-resolution description of the order of main events driving G-protein activation in response to GTP binding. Structural changes propagate from the nucleotide-binding pocket and extend through the GTPase domain, enacting alterations to Gα switch regions and the α5 helix that weaken the G-protein-receptor interface. Molecular dynamics simulations with late structures in the cryo-EM trajectory support that enhanced ordering of GTP on closure of the α-helical domain against the nucleotide-bound Ras-homology domain correlates with α5 helix destabilization and eventual dissociation of the G protein from the GPCR. These findings also highlight the potential of time-resolved cryo-EM as a tool for mechanistic dissection of GPCR signalling events.
Assuntos
Microscopia Crioeletrônica , Subunidades alfa Gs de Proteínas de Ligação ao GTP , Receptores Adrenérgicos beta 2 , Humanos , Sítios de Ligação , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/efeitos dos fármacos , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/ultraestrutura , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacologia , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/ultraestrutura , Fatores de Tempo , Ativação Enzimática/efeitos dos fármacos , Domínios Proteicos , Estrutura Secundária de Proteína , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Neoplasias Cerebelares , Cromograninas , Subunidades alfa Gs de Proteínas de Ligação ao GTP , Meduloblastoma , Ossificação Heterotópica , Dermatopatias Genéticas , Humanos , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Masculino , Cromograninas/genética , Meduloblastoma/genética , Meduloblastoma/patologia , Ossificação Heterotópica/genética , Ossificação Heterotópica/patologia , Dermatopatias Genéticas/genética , Dermatopatias Genéticas/patologia , Dermatopatias Genéticas/complicações , Lactente , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Neoplasias Cerebelares/complicações , Prognóstico , Doenças Ósseas Metabólicas/genética , Doenças Ósseas Metabólicas/patologia , MutaçãoRESUMO
BACKGROUND: Indistinct and analogous histopathological features of various fibro-osseous lesions make establishing a definitive diagnosis a challenge. There is a need for additional molecular and histochemical tools to support and differentiate these lesions in order to establish a concrete diagnosis. MATERIALS AND METHODS: A retrospective analysis of biopsied lesions in formalin-fixed paraffin-embedded sections (10 cases each of fibrous dysplasia, ossifying fibroma, and cement-osseous dysplasia) retrieved from the archives was studied for immunoexpression of osteocalcin (quantitative analysis in osteocytes), collagen characterization using Azan, Picrosirus, and Toluidine blue stain for evaluating intensity and localization of collagen fibers, and morphometric analysis of vasculature (for evaluating mean vessel density as square microns). RESULTS: Positive immunostaining of osteocalcin suggested mutations of the GNAS-1 gene found in fibrous dysplasia indirectly, as it is a negative regulator of bone formation. Osteocalcin immunopositivity was quantitatively measured in the fibro-osseous lesions, with fibrous dysplasia measuring 14.47 ± 3.628 as compared to ossifying fibroma measuring 5.23 ± 1.33, followed by cemento-osseous dysplasia measuring 2.30 ± 1.409. Toluidine blue suggests the presence of oxytalan fibers (resistant to acid hydrolysis) in ossifying fibroma and cemento-osseous dysplasia, pointing toward the pathogenesis of the lesion. Azan stain and Picrosirus (under a polarizing microscope) helped in distinguishing hard tissue characteristics (70% of cases of fibrous dysplasia showed only a magenta component followed by intermixed magenta with a blue component in 20% of cases and only 10% of cases showed magenta with blue borders whereas for ossifying fibroma, 40% of cases depicted magenta with blue borders along with the other 40% with intermixed magenta with blue component). The mean vessel density was also highest in fibrous dysplasia measuring 7.90 ± 1.079 (in Sq. micron area), followed by ossifying fibroma and cemento-osseous dysplasia. CONCLUSION: The diagnosis of fibro-osseous lesions by hematoxylin and eosin alone is confusing and thus should be supported by relatively simple histomorphometric analysis for better treatment outcomes. At the diagnostic stage of fibro-osseous lesions, evaluation of intralesional vessel size, reliable molecular marker, and histochemical nature can aid in differentiating fibrous dysplasia from central ossifying fibroma and cemento-osseous dysplasia alongside, other clinical, radiographic and pathological criteria. These parameters help in the diagnostic decision-making of fibro-osseous lesions.
Assuntos
Fibroma Ossificante , Displasia Fibrosa Óssea , Imuno-Histoquímica , Osteocalcina , Humanos , Estudos Retrospectivos , Fibroma Ossificante/patologia , Fibroma Ossificante/genética , Fibroma Ossificante/diagnóstico , Displasia Fibrosa Óssea/genética , Displasia Fibrosa Óssea/patologia , Displasia Fibrosa Óssea/diagnóstico , Osteocalcina/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Cromograninas/genética , Cementoma/patologia , Cementoma/diagnóstico , Cementoma/genética , Colágeno , Masculino , Feminino , BiópsiaRESUMO
IMPLICATIONS: The study identifies an opportunity to discover a PKA-independent pathway downstream of oncogene GNAS for managing IPMN lesions and their progression to PDAC.
Assuntos
Proliferação de Células , Cromograninas , Proteínas Quinases Dependentes de AMP Cíclico , Subunidades alfa Gs de Proteínas de Ligação ao GTP , Organoides , Neoplasias Pancreáticas , Humanos , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Organoides/metabolismo , Organoides/patologia , Cromograninas/genética , Cromograninas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Ductos Pancreáticos/metabolismo , Ductos Pancreáticos/patologia , Ductos Pancreáticos/citologia , Células Acinares/metabolismo , Células Acinares/patologiaRESUMO
G proteins are interacting partners of G protein-coupled receptors (GPCRs) in eukaryotic cells. Upon G protein activation, the ability of the Gα subunit to exchange GDP for GTP determines the intracellular signal transduction. Although various studies have successfully shown that both Gαs and Gαi have an opposite effect on the intracellular cAMP production, with the latter being commonly described as "more active", the functional analysis of Gαs is a comparably more complicated matter. Additionally, the thorough investigation of the ubiquitously expressed variants of Gαs, Gαs(short) and Gαs(long), is still pending. Since the previous experimental evaluation of the activity and function of the Gαs isoforms is not consistent, the focus was laid on structural investigations to understand the GTPase activity. Herein, we examined recombinant human Gαs by applying an established methodological setup developed for Gαi characterization. The ability for GTP binding was evaluated with fluorescence and fluorescence anisotropy assays, whereas the intrinsic hydrolytic activity of the isoforms was determined by a GTPase assay. Among different nucleotide probes, BODIPY FL GTPγS exhibited the highest binding affinity towards the Gαs subunit. This work provides a deeper understanding of the Gαs subunit and provides novel information concerning the differences between the two protein variants.
Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP , Humanos , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Nucleotídeos de Guanina/metabolismo , Nucleotídeos de Guanina/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Guanosina Trifosfato/metabolismoRESUMO
Approximately 5% of colorectal cancers (CRCs) have a gain-of-function mutation in the GNAS gene, which leads to the activation of cAMP-dependent signaling pathways and associates with poor prognosis. We investigated the effect of an activating GNAS mutation in CRC cell lines on gene expression and cell proliferation in vitro, and tumor growth in vivo. GNAS-mutated (GNASmt) HCT116 cells showed stimulated synthesis of cAMP as compared to parental (Par) cells. The most upregulated gene in the GNASmt cells was cAMP-hydrolyzing phosphodiesterase 4D (PDE4D) as detected by RNA sequencing. To further validate our finding, we analyzed PDE4D expression in a set of human CRC tumors (n = 35) and demonstrated overexpression in GNAS mutant CRC tumors as compared to GNAS wild-type tumors. The GNASmt HCT116 cells proliferated more slowly than the Par cells. PDE4 inhibitor Ro 20-1724 and PDE4D subtype selective inhibitor GEBR-7b further suppressed the proliferation of GNASmt cells without an effect on Par cells. The growth inhibitory effect of these inhibitors was also seen in the intrinsically GNAS-mutated SK-CO-1 CRC cell line having high levels of cAMP synthesis and PDE4D expression. In vivo, GNASmt HCT116 cells formed smaller tumors than the Par cells in nude mice. In conclusion, our findings demonstrate that GNAS mutation results in the growth suppression of CRC cells. Moreover, the GNAS mutation-induced overexpression of PDE4D provides a potential avenue to impede the proliferation of CRC cells through the use of PDE4 inhibitors.