Guanine Nucleotide Binding Protein Alpha Research Articles

PRKACB is a novel imprinted gene in marsupials

BackgroundGenomic imprinting results in parent-of-origin-specific gene expression and, among vertebrates, is found only in therian mammals: marsupials and eutherians. A differentially methylated region (DMR), in which the methylation status of CpG dinucleotides differs between the two alleles, can mark the parental identity of imprinted genes. We developed a computational pipeline that detected CpG islands (CGIs) marked by both methylated and unmethylated signals in whole genome bisulfite sequencing data. This approach identified candidate marsupial DMRs in a publicly available koala methylome. One of these candidate DMRs was associated with PRKACB, a gene encoding the protein kinase A catalytic subunit beta. Nothing is known about the imprinting status of PRKACB in eutherian mammals although mutations of this gene are associated with endocrine neoplasia and other developmental disorders.ResultsIn the tammar wallaby and brushtail possum there was parent-of-origin-specific DNA methylation in the PRKACB DMR in which the maternal allele was methylated and the paternal allele was unmethylated. There were multiple RNAs transcribed from this locus. Allele-specific expression analysis identified paternal expression of a PRKACB lncRNA and an mRNA isoform. Comparison of the PRKACB gene start site between marsupials and eutherians demonstrated that the CGI is longer in marsupials. The PRKACB gene product functions in the same signalling pathway as the guanine nucleotide-binding protein alpha subunit encoded at the GNAS locus, a known eutherian imprinted gene. In a mouse methylome Gnas had three differentially methylated CGIs, while in the koala methylome the GNAS locus had two unmethylated CGIs.ConclusionsWe conclude that PRKACB is a novel, DMR-associated marsupial imprinted gene. Imprinting of PRKACB in marsupials and GNAS in eutherians may indicate a conserved selection pressure for imprinting of the protein kinase A signalling pathway in therians with the two lineages adapting by imprinting different genes.

Progress on immuno-microenvironment and immune-related therapies in patients with pseudomyxoma peritonei.

Pseudomyxoma peritonei (PMP) is an indolent malignant syndrome. The standard treatment for PMP is cytoreductive surgery combined with intraperitoneal hyperthermic chemotherapy (CRS + HIPEC). However, the high recurrence rate and latent clinical symptoms and signs are major obstacles to further improving clinical outcomes. Moreover, patients in advanced stages receive little benefit from CRS + HIPEC due to widespread intraperitoneal metastases. Another challenge in PMP treatment involves the progressive sclerosis of PMP cell-secreted mucus, which is often increased due to activating mutations in the gene coding for guanine nucleotide-binding protein alpha subunit (GNAS). Consequently, the development of other PMP therapies is urgently needed. Several immune-related therapies have shown promise, including the use of bacterium-derived non-specific immunogenic agents, radio-immunotherapeutic agents, and tumor cell-derived neoantigens, but a well-recognized immunotherapy has not been established. In this review the roles of GNAS mutations in the promotion of mucin secretion and disease development are discussed. In addition, the immunologic features of the PMP microenvironment and immune-associated treatments are discussed to summarize the current understanding of key features of the disease and to facilitate the development of immunotherapies.

Effect of GNA13 on the alteration of tumor microenvironmental components through exosomes to promote the development of head and neck tumors.

e18035 Background: Guanine nucleotide-binding protein alpha subunit 13 (GNA13) is closely related to tumor development, but the effect of GNA13 on the biological behavior of head and neck tumors is unknown. The aim of this study was to investigate whether GNA13 can influence the alteration of tumor microenvironment components through exosomes, which in turn promotes the development of head and neck squamous carcinoma (HNSCC). Methods: Using the Gene Expression Omnibus database, the differentially expressed gene GNA13 was screened. Immunohistochemistry and western blotting were used to detect the expression of GNA13 in HNSCC tissues and control tissues. GNA13 expression was down-regulated in tumor cell lines using transfection techniques. The effect of GNA13 downregulation on tumor cell growth was examined by in vitro experiments. RT-PCR and dual luciferase reporter gene assays were used to validate GNA13-regulated miRNAs and their target genes. Clonogenesis assays were used to examine the effect of target gene expression on fibroblasts, and the HNSCC xenograft mouse model was used to verify the role of GNA13 in tumor growth in vivo. The peripheral blood from patients with different stages of HNSCC was collected to determine the expression level of GNA13 in serum exosomes and to analyze its relationship with metastasis and postoperative recurrence. Results: GNA13 expression was elevated in HNSCC tissues and cells. The growth capacity of HNSCC cells was significantly reduced after downregulation of GNA13, which induced fibroblast conversion to MAF via exosomes and promoted their proliferation and migration. Meanwhile, GNA13 was able to regulate miR-26a-5p/STRADB expression in fibroblasts via exosomes. The overexpression of STRADB promoted the proliferation and migration of fibroblasts and induced their conversion to MAF. More importantly, in the conditioned medium of STRADB overexpressing fibroblasts, the proliferation and migration of HNSCC cells were promoted. Furthermore, knockdown of GNA13 expression inhibited the tumor growth in nude mice. It appeared that high expression of GNA13 in serum exosomes of patients with HNSCC was associated with poor prognosis. Conclusions: GNA13 regulates the miR-26a-5p/STRADB axis in fibroblasts via exosomes to create a metastasis-friendly microenvironment for tumor cells, ultimately promoting the progression of HNSCC. Meanwhile, the expression of GNA13 was positively correlated with the pathological grade and clinical stage of HNSCC, which seems to be a reliable indicator of tumor recurrence. Targeting this signaling pathway would be a promising therapeutic strategy for HNSCC.

Circ_0000595 knockdown alleviates CoCl2-mediated effects in VSMCs by regulating the miR-582-3p/ADAM10 axis.

Thoracic aortic aneurysm (TAA) is a serious vascular disease causing the death of elder people. Accumulating studies have reported that circular RNAs (circRNAs) are implicated in the regulation of aortic aneurysms. However, the role of circ_0000595 in the progression of TAA is still unclear. Quantitative real-time PCR (qRT-PCR) and western blotting were implemented to assess circ_0000595, microRNA (miR)-582-3p, guanine nucleotide-binding protein alpha subunit (ADAM10), PCNA, Bax, and Bcl-2 expression. The proliferation of vascular smooth muscle cells was determined using cell counting kit 8 (CCK-8) and 5-ethynyl-2-deoxyuridine (EdU). Cell apoptosis was measured using flow cytometry, and caspase-3 activity was analyzed using a commercial kit. After bioinformatics analysis, the interaction between miR-582-3p and circ_0000595 or ADAM10 was validated using a dual-luciferase reporter and RNA immunoprecipitation. As compared with controls, TAA tissues and CoCl2-induced VSMCs displayed high expression of circ_0000595 and ADAM10, and low expression of miR-582-3p. CoCl2 treatment evidently suppressed VSMC proliferation and promoted VSMCs apoptosis, and these impacts were reverted by circ_0000595 knockdown. Circ_0000595 acted as a molecular sponge for miR-582-3p, and circ_0000595 silencing-mediated influences in CoCl2-induced VSMCs were overturned by miR-582-3p inhibitor. ADAM10 was confirmed as a target gene of miR-582-3p, and miR-582-3p overexpression-induced influence was almost restored by overexpressed ADAM10 in CoCl2-induced VSMCs. Besides, circ_0000595 contributed to ADAM10 protein expression by sponging miR-582-3p. Our data verified that circ_0000595 silencing might attenuate CoCl2-mediated impacts in VSMCs by regulating the miR-582-3p/ADAM10 axis, providing new potential roads for treating TAA.

Detrimental Role of PDZ-RhoGEF in Pathological Cardiac Hypertrophy.

Postsynaptic density 95/disk-large/ZO-1 Rho guanine nucleotide exchange factor (PDZ-RhoGEF, PRG) functions as a RhoGEF for activated Gα13 and transmits activation signals to downstream signaling pathways in various pathological processes. Although the prohypertrophic effect of activated Gα13 (guanine nucleotide binding protein alpha 13; a heterotrimeric G protein) is well-established, the role of PDZ-RhoGEF in pathological cardiac hypertrophy is still obscure. Genetically engineered mice and neonatal rat ventricular myocytes were generated to investigate the function of PRG in pathological myocardial hypertrophy. The prohypertrophic stimuli-induced alternations in the morphology and intracellular signaling were measured in myocardium and neonatal rat ventricular myocytes. Furthermore, multiple molecular methodologies were used to identify the precise molecular mechanisms underlying PDZ-RhoGEF function. Increased PDZ-RhoGEF expression was documented in both hypertrophied hearts and neonatal rat ventricular myocytes. Upon prohypertrophic stimuli, the PDZ-RhoGEF-deficient hearts displayed alleviated cardiomyocyte enlargement and attenuated collagen deposition with improved cardiac function, whereas the adverse hypertrophic responses in hearts and neonatal rat ventricular myocytes were markedly exaggerated by PDZ-RhoGEF overexpression. Mechanistically, RhoA (ras homolog family member A)-dependent signaling pathways may function as the downstream effectors of PDZ-RhoGEF in hypertrophic remodeling, as confirmed by rescue experiments using a RhoA inhibitor and dominant-negative RhoA. Furthermore, PDZ-RhoGEF is associated with activated Gα13 and contributes to Gα13-mediated activation of RhoA-dependent signaling. Our data provide the first evidence that PDZ-RhoGEF promotes pathological cardiac hypertrophy by linking activated Gα13 to RhoA-dependent signaling pathways. Therefore, PDZ-RhoGEF has the potential to be a diagnostic marker or therapeutic target for pathological cardiac hypertrophy.

GNAQ mutations drive port wine birthmark-associated Sturge-Weber syndrome: A review of pathobiology, therapies, and current models.

Port-wine birthmarks (PWBs) are caused by somatic, mosaic mutations in the G protein guanine nucleotide binding protein alpha subunit q (GNAQ) and are characterized by the formation of dilated, dysfunctional blood vessels in the dermis, eyes, and/or brain. Cutaneous PWBs can be treated by current dermatologic therapy, like laser intervention, to lighten the lesions and diminish nodules that occur in the lesion. Involvement of the eyes and/or brain can result in serious complications and this variation is termed Sturge-Weber syndrome (SWS). Some of the biggest hurdles preventing development of new therapeutics are unanswered questions regarding disease biology and lack of models for drug screening. In this review, we discuss the current understanding of GNAQ signaling, the standard of care for patients, overlap with other GNAQ-associated or phenotypically similar diseases, as well as deficiencies in current in vivo and in vitro vascular malformation models.

Circ_0004491 stimulates guanine nucleotide-binding protein alpha subunit to inhibit the malignant progression of oral squamous cell carcinoma by sponging miR-2278

Background/purposeCircular RNA origin recognition complex subunit 4 (circORC4; ID: hsa_circ_0004491) have been confirmed to be a novel potential biomarker of oral squamous cell carcinoma (OSCC). This study aimed to explore the molecular mechanism of circ_0004491 in OSCC progression. Materials and methodsLevels of circ_0004491, microRNA (miR)-2278, guanine nucleotide-binding protein alpha subunit (GNAS), Bax, Bcl-2, E-cadherin and ki-67 were detected by quantitative real-time PCR, western blotting and immunohistochemistry. The proliferation of OSCC cells was measured using colony formation assay and EdU staining. Cell apoptosis and motility were detected by flow cytometry and transwell assays respectively. Interaction between miR-2278 and circ_0004491 or GNAS was predicted by bioinformatics analysis and confirmed via luciferase reporter assay and RNA immunoprecipitation assay. Xenograft tumor model was used to analyze the role of circ_0004491 in tumor growth in vivo. ResultsCirc_0004491 was downregulated in OSCC tissues and cell lines. Circ_0004491 overexpression suppressed the proliferation, migration and invasion whereas facilitated the apoptosis of OSCC cells. Circ_0004491 acted as a molecular sponge for miR-2278, and circ_0004491 overexpression-mediated effect was partly reversed by miR-2278 mimic in OSCC cells. MiR-2278 interacted with the 3’UTR of GNAS. Circ_0004491 contributed to GNAS level by sponging miR-2278 in OSCC cells. GNAS knockdown restored miR-2278 inhibitor-mediated effect in OSCC cells. Circ_0004491 overexpression repressed xenograft tumor growth in vivo. ConclusionCirc_0004491 can repress OSCC progression by regulation of miR-2278/GNAS axis, providing a possible circRNA-targeted therapy for OSCC.

Overexpression of GNA13 correlates with poor prognosis in esophageal squamous cell carcinoma after esophagectomy.

This study aimed to explore the expression and clinical implication of guanine nucleotide-binding protein alpha 13 (GNA13) in esophageal squamous cell carcinoma (ESCC). We first employed western blot analysis to test the GNA13 protein expression level in ESCC tissues. Subsequently, we used immunohistochemistry assays to detect the GNA13 in ESCC specimens from 173 patients who underwent esophagectomy. Survival analysis was performed to define the impact of GNA13 expressions on the prognosis of the ESCC patients based on the clinical and follow-up data. The GNA13 protein was shown to be considerably higher in ESCC tissues than in normal esophageal tissues. The level of expression was closely related to the tumor, node, TNM stage, and tumor size. More importantly, ESCC patients with high GNA13 expression carried an increased risk of tumor recurrence compared to those with low GNA13 expression. In addition, a high GNA13 expression level could independently predict worse overall survival and disease-free survival in ESCC. GNA13 could be a novel prognostic biomarker for ESCC patients after esophagectomy.

Sex and brain region-specific regulation of serotonin transporter activity in synaptosomes in guanine nucleotide-binding protein G(q) alpha knockout mice.

The regulation of the serotonin transporter (SERT) by guanine nucleotide-binding protein alpha (Gα) q was investigated using Gαq knockout mice. In the absence of Gαq, SERT-mediated uptake of 5-hydroxytryptamine (5HT) was enhanced in midbrain and frontal cortex synaptosomes, but only in female mice. The mechanisms underlying this sexual dimorphism were investigated using quantitative western blot analysis revealing brain region-specific differences. In the frontal cortex, SERT protein expression was decreased in male knockout mice, seemingly explaining the sex-dependent variation in SERT activity. The differential expression of Gαi1 in female mice contributes to the sex differences in the midbrain. In fact, Gαi1 levels inversely correlate with 5HT uptake rates across both sexes and genotypes. Likely due to differential SERT regulation as well as sex differences in the expression of tryptophan hydroxylase 2, Gαq knockout mice also displayed sex- and genotype-dependent alterations in total 5HT tissue levels as determined by high-performance liquid chromatography. Gαq inhibitors, YM-254890 and BIM-46187, differentially affected SERT activity in both, synaptosomes and cultured cells. YM-254890 treatment mimicked the effect of Gαq knockout in the frontal cortex. BIM-46187, which promotes the nucleotide-free form of Gα proteins, substantially inhibited 5HT uptake, prompting us to hypothesise that Gαq interacts with SERT similarly as with G-protein-coupled receptors and inhibits SERT activity by modulating transport-associated conformational changes. Taken together, our findings reveal a novel mechanism of SERT regulation and impact our understanding of sex differences in diseases associated with dysregulation of serotonin transmission, such as depression and anxiety.

A novel GNAS mutation in pseudohypoparathyroidism type 1a in a Chinese man presented with recurrent seizure: a case report

BackgroundPseudohypoparathyroidism is a rare genetic disease characterized by hypocalcaemia and hyperphosphataemia due to the defect to the guanine nucleotide-binding protein alpha subunit (GNAS) gene. Patients with pseudoparathyroidism type 1a and 1c could manifest Albright’s hereditary osteodystrophy and multiple hormone resistance including gonadotropin and thyroid stimulating hormone.Case presentationHere we report a Chinese man who presented with fatigue, recurrent seizure and Albright’s hereditary osteodystrophy. His genetic study revealed a heterozygote mutation in the GNAS gene [NM_000516.4(GNAS): c2787_2788del (p.Val930AspfsTer12)]. After calcium and calcitriol supplement, his seizures achieved partially remission.ConclusionsWe report a case of PHP1a or 1c with a novel frameshift mutation in GNAS gene in a patient presenting with AHO, as well as TSH and partial gonadotropin resistance. This mutation in this case has not been reported in literature and adds to the spectrum of genetic mutations related to PHP.

A novel GNAS-mutated human induced pluripotent stem cell model for understanding GNAS-mutated tumors.

A missense mutation of the guanine nucleotide binding protein alpha stimulating activity polypeptide 1 (GNAS) gene, typically Arg201Cys or Arg201His (R201H/R201C), leads to constitutive activation of the Gsα-cyclic AMP (cAMP) signaling pathway that causes several diseases. However, no germline mutations of GNAS have been identified to date, likely due to their lethality, and no robust human cell models have been generated. Therefore, the aim of this study was to generate GNAS-mutated disease-specific induced pluripotent stem cells as a model for these diseases. We then analyzed the functionality of this induced pluripotent stem cell model and differentiated epithelial cells. We generated disease-specific induced pluripotent stem cells by introducing a mutation in GNAS with the clustered regularly interspaced short palindromic repeats (CRISPR) nickase method, which has lower off-target effects than the conventional CRISPR/Cas9 method. We designed the target vector to contain the R201H mutation in GNAS, which was transfected into human control induced pluripotent stem cells (Nips-B2) by electroporation. We confirmed the establishment of GNASR201H-mutated (GNASR201H/+) induced pluripotent stem cells that exhibited a pluripotent stem cell phenotype. We analyzed the effect of the mutation on cAMP production, and further generated teratomas for immunohistochemical analysis of the luminal epithelial structure. GNAS-mutated induced pluripotent stem cells showed significantly higher levels of intracellular cAMP, which remained elevated state for a long time upon hormonal stimulation with parathyroid hormone or adrenocorticotropic hormone. Immunohistochemical analysis revealed that several mucins, including MUC1, 2, and MUC5AC, are expressed in cytokeratin 18 (CK18)-positive epithelial cells. However, we found few CK18-positive cells in mutated induced pluripotent stem cell-derived teratoma tissues, and reduced MUCINs expression in mutated epithelial cells. There was no difference in CDX2 expression; however, mutated epithelial cells were positive for CEA and CA19-9 expression. GNASR201H-mutated induced pluripotent stem cells and GNASR201H-mutated epithelial cells have distinct phenotypic and differentiation characteristics. We successfully established GNASR201H-mutated human induced pluripotent stem cells with increased cAMP production. Considering the differentiation potential of induced pluripotent stem cells, these cells will be useful as a model for elucidating the pathological mechanisms of GNAS-mutated diseases.

RNA-seq Profiling and Co-expression Network Analysis of Long Noncoding RNAs and mRNAs Reveal Novel Pathogenesis of Noise-induced Hidden Hearing Loss

Noise-induced hidden hearing loss (NIHHL), one of the family of conditions described as noise-induced hearing loss (NIHL), is characterized by synaptopathy following moderate noise exposure that causes only temporary threshold elevation. Long noncoding RNAs (lncRNAs) mediate several essential regulatory functions in a wide range of biological processes and diseases, but their roles in NIHHL remain largely unknown. In order to determine the potential roles of these lncRNAs in the pathogenesis of NIHHL, we first evaluated their expression in NIHHL mice model and mapped possible regulatory functions and targets using RNA-sequencing (RNA-seq). In total, we identified 133 lncRNAs and 522 mRNAs that were significantly dysregulated in the NIHHL model. Gene Ontology (GO) showed that these lncRNAs were involved in multiple cell components and systems including synapses and the nervous and sensory systems. In addition, a lncRNA–mRNA network was constructed to identify core regulatory lncRNAs and transcription factors. KEGG analysis was also used to identify the potential pathways being affected in NIHHL. These analyses allowed us to identify the guanine nucleotide binding protein alpha stimulating (GNAS) gene as a key transcription factor and the adrenergic signaling pathway as a key pathway in the regulation of NIHHL pathogenesis. Our study is the first, to our knowledge, to isolate a lncRNA mediated regulatory pathway associated with NIHHL pathogenesis; these observations may provide fresh insight into the pathogenesis of NIHHL and may pave the way for therapeutic intervention in the future.

Hypothalamic Paraventricular Nucleus Gαi2 (Guanine Nucleotide-Binding Protein Alpha Inhibiting Activity Polypeptide 2) Protein-Mediated Neural Control of the Kidney and the Salt Sensitivity of Blood Pressure.

We have previously reported that in salt-resistant rat phenotypes brain, Gαi2 (guanine nucleotide-binding protein alpha inhibiting activity polypeptide 2) proteins are required to maintain blood pressure and sodium balance. However, the impact of hypothalamic paraventricular nucleus (PVN) Gαi2 proteins on the salt sensitivity of blood pressure is unknown. Here, by the bilateral PVN administration of a targeted Gαi2 oligodeoxynucleotide, we show that PVN-specific Gαi2 proteins are required to facilitate the full natriuretic response to an acute volume expansion (peak natriuresis [μeq/min] scrambled (SCR) oligodeoxynucleotide 41±3 versus Gαi2 oligodeoxynucleotide 18±4; P<0.05) via a renal nerve-dependent mechanism. Furthermore, in response to chronically elevated dietary sodium intake, PVN-specific Gαi2 proteins are essential to counter renal nerve-dependent salt-sensitive hypertension (mean arterial pressure [mm Hg] 8% NaCl; SCR oligodeoxynucleotide 128±2 versus Gαi2 oligodeoxynucleotide 147±3; P<0.05). This protective pathway involves activation of PVN Gαi2 signaling pathways, which mediate sympathoinhibition to the blood vessels and kidneys (renal norepinephrine [pg/mg] 8% NaCl; SCR oligodeoxynucleotide 375±39 versus Gαi2 oligodeoxynucleotide 850±27; P<0.05) and suppression of the activity of the sodium chloride cotransporter assessed as peak natriuresis to hydrochlorothiazide. Additionally, central oligodeoxynucleotide-mediated Gαi2 protein downregulation prevented PVN parvocellular neuron activation, assessed by FosB immunohistochemistry, in response to increased dietary salt intake. In our analysis of the UK BioBank data set, it was observed that 2 GNAI2 single nucleotide polymorphism (SNP) (rs2298952, P=0.041; rs4547694, P=0.017) significantly correlate with essential hypertension. Collectively, our data suggest that selective targeting and activation of PVN Gαi2 proteins is a novel therapeutic approach for the treatment of salt-sensitive hypertension.

Post Traumatic Fibrous Dysplasia of Ribs at the Chest Tube Site (A Case Report)

Fibrous Dysplasia (FD) is a benign non-inherited and rare skeletal disorder that can affect any bone of human body. FD is caused by a mutation in GNAS1 gene, but the mutation is not inherited from parents. While there are some reports of post-traumatic fibrous dysplasia, but the relationship between fibrous dysplasia and trauma or previous injury is unclear. Most of the time, fibrous dysplasia is diagnosed by accident in x-ray imaging. While there is much information on fibrous dysplasia, the main etiology is still unknown, however it seems to be linked to a gene mutation. A mutation in this gene affects differentiation and proliferation of cells. This benign fibro-osseous lesion is a result of disturbances of normal bone metabolism by a mutation of the alpha subunit of guanine nucleotide binding protein alpha stimulating. Here we report a case with two adjacent ribs involvement in a 25-year old man who admitted to our hospital with an acute right chest pain and swelling with history of major trauma in a motor vehicle accident 4 years ago with associated hemo-pneumothorax resulted in thoracostomy tube insertion and with subcutaneous mass was detected in the right chest wall which was located exactly at the thoracostomy tube insertion site.

WITHDRAWN: Comprehensive analysis of gene mutation and expression profiles in fibrous dysplasia of bone via next-generation sequencing

// Hongbo He 1 , Qing Liu 1 , Can Zhang 1 , Zhan Liao 1 , Yupeng Liu 1 , Jun Wan 1 , Jian Tian 1 , Xiaopeng Tong 1 , Lichun Sun 1,2,3 and Wei Luo 1 1 Department of Orthopedics, Xiangya Hospital, Central South University, Changsha, Hunan, China 2 Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study, Institute of Pharmacy & Pharmacology, University of South China, Hengyang, China 3 Department of Medicine, School of Medicine, Tulane Health Sciences Center, New Orleans, LA, USA Correspondence to: Wei Luo, email: luowei0928@126.com Keywords : fibrous dysplasia; whole-exome sequencing (WES); transcriptome sequencing (RNAseq); single gene sequencing; SNV/InDel Received: May 07, 2017 Accepted: November 13, 2017 Epub: January 03, 2018 Abstract Fibrous dysplasia (FD) of bone is a rare and non-inheritable bone disease. Its precise molecular mechanisms remain unclear. We collected FD patient samples and applied the innovative technology of whole-exome sequencing (WES) and transcriptome sequencing (RNAseq) to analyze the gene mutations and expression profiles. By comparing gene mutations with both WES and RNAseq, and further filtering these mutations with gene expressional significance (average > 1TPM), we eventually identified 20 genes with non-synonymous single nucleotide variations (SNVs) occurring in the exonic regions of all FD samples. About 40 genes with SNVs were mutated in 50% of the FD samples, including GNAS (Guanine Nucleotide Binding Protein Alpha) and RTN4 (Reticulon 4). Particularly, in three of these patients (Patient 1, 3 and 4), GNAS were identified as having SNVs, with the nucleotide changed in exon 8 and the amino acid residue changed in position 201 (GNAS: NM_000516: exon8: c.G602A: p.R201H; or c.C601T: p.R201C). We only observed 2 genes, FAM21B (mutation type: deletion) and GAGE2A (mutation type: amplification), with copy number variations (CNVs) occurred in FD patients. When analyzing signaling pathways and biological functions, we predicted some key genes, such as GPCRs, G proteins, chemokine and ANK-associated genes, and their associated signaling networks might play critical roles in FD development. We further confirmed these observations via single gene sequencing and Q-PCR. Thus, the mutations of these genes may be connected with fibroblast and bone development disorders. These findings may provide a clue for our next investigation of FD pathogenesis and its molecular mechanisms.

Expression and localization of guanine nucleotide-binding protein alpha S in the testis and epididymis of rams at different developmental stages

The guanine nucleotide-binding alpha S subunit (Gαs) is an important element of key signaling pathways, which is widely expressed in mammalian tissues; however, its role in the reproductive system is still unclear. In this study, we investigated the expression and localization of Gαs in the testes and epididymis of rams at different developmental stages using quantitative RT-PCR, immunohistochemistry, and western blotting. In 1-, 6-, and 12-month-old rams, the transcription of Gαs-encoding gene (Gnαs) was significantly upregulated in the corpus and cauda epididymis compared to the testes and caput epididymis (P<0.05). At 12 months, the level of Gnαs mRNA was higher than that at 1 and 6 months for all tested tissues (P<0.05). The Gαs protein was detected in the principal cells and interstitial epididymal cells, including Sertoli and Leydig cells, as well as in testicular cells, spermatogonial stem cells, and spermatocytes. Gαs expression was the highest in the cauda epididymis (P<0.05), followed by the corpus epididymis, caput epididymis, and testes. The results indicate that in the reproductive organs of rams, Gαs is expressed in a tissue-specific and age-dependent manner. The high levels of Gαs observed in the epididymis suggest that Gαs may influence the composition of the epididymal lumen fluid and, consequently, the microenvironment for spermatozoa maturation. Thus, Gαs could play an important role in spermatogenesis and the development of the testes and epididymis in the reproductive system of rams.

PSEUDOHYPOPARATHYROIDISM Ia TYPE WITH EARLY DEBUT IN SISTERS OF ONE FAMILY

Pseudohypoparathyroidism is a rare genetic disorder characterised by end-organ resistance to parathyroid hormone due to a defect of the guanine nucleotide-binding protein alpha that simulates activity of the polypeptide 1 (GNAS) gene. Patients with type Ia pseudohypoparathyroidism have different features of Albright's hereditary osteodystrophy and characteristic phenotype (obesity, round face, short stature, short neck, brachidactyly, etc.), multi-hormone resistance. We describe two sisters (half sibs), who presented with different symptoms of pseudohypoparathyroidism and clinically manifested different degree of Albright's hereditary osteodystrophy. Genetic study detected a mutation p.D190MfsX14 (c.568 571 delGACT), in theGNAS 1 gene (OMIM*139320).

Valproic acid mediates miR-124 to down-regulate a novel protein target, GNAI1

Valproic acid (VPA) is an anti-convulsant drug that is recently shown to have neuroregenerative therapeutic actions. In this study, we investigate the underlying molecular mechanism of VPA and its effects on Bdnf transcription through microRNAs (miRNAs) and their corresponding target proteins. Using in silico algorithms, we predicted from our miRNA microarray and iTRAQ data that miR-124 is likely to target at guanine nucleotide binding protein alpha inhibitor 1 (GNAI1), an adenylate cyclase inhibitor. With the reduction of GNAI1 mediated by VPA, the cAMP is enhanced to increase Bdnf expression. The levels of GNAI1 protein and Bdnf mRNA can be manipulated with either miR-124 mimic or inhibitor. In summary, we have identified a novel molecular mechanism of VPA that induces miR-124 to repress GNAI1. The implication of miR-124→GNAI1→BDNF pathway with valproic acid treatment suggests that we could repurpose an old drug, valproic acid, as a clinical application to elevate neurotrophin levels in treating neurodegenerative diseases.

Advances in genetic study of achromatopsia

Achromatopsia is a kind of autosomal recessive cone disorder. It occurs with nystagmus, photophobia, inability of color discrimination and severely reduced visual acuity. Five pathogenic genes had been reported to be associated with achromatopsia: cyclic nucleotide-gated(CNG)A3, CNGB3, guanine nucleotide binding protein alpha transduction active pepitide 2(GNAT2), phosphodiesterase (PDE)6C and PDE6H. They are crucial for cone phototransduction. Mutations of these genes can induce achromatopsia. Gene therapy, which can recover partial visual function, has been successfully used for the treatment of achromatopsia in animal model. Clinical features, pathogenic genes functions and mutations, the animal models and gene therapy of achromatopsia were reviewed. Key words: Achromatopsia; Mutation; Disease models, animal; Gene therapy

Mutation analysis of inhibitory guanine nucleotide binding protein alpha (GNAI) loci in young and familial pituitary adenomas.

Pituitary adenomas are neoplasms of the anterior pituitary lobe and account for 15–20% of all intracranial tumors. Although most pituitary tumors are benign they can cause severe symptoms related to tumor size as well as hypopituitarism and/or hypersecretion of one or more pituitary hormones. Most pituitary adenomas are sporadic, but it has been estimated that 5% of patients have a familial background. Germline mutations of the tumor suppressor gene aryl hydrocarbon receptor-interacting protein (AIP) predispose to hereditary pituitary neoplasia. Recently, it has been demonstrated that AIP mutations predispose to pituitary tumorigenesis through defective inhibitory GTP binding protein (Gαi) signaling. This finding prompted us to examine whether germline loss-of-function mutations in inhibitory guanine nucleotide (GTP) binding protein alpha (GNAI) loci are involved in genetic predisposition of pituitary tumors. To our knowledge, this is the first time GNAI genes are sequenced in order to examine the occurrence of inactivating germline mutations. Thus far, only somatic gain-of-function hot-spot mutations have been studied in these loci. Here, we have analyzed the coding regions of GNAI1 , GNAI2, and GNAI3 in a set of young sporadic somatotropinoma patients (n = 32; mean age of diagnosis 32 years) and familial index cases (n = 14), thus in patients with a disease phenotype similar to that observed in AIP mutation carriers. In addition, expression of Gαi proteins was studied in human growth hormone (GH), prolactin (PRL), adrenocorticotropic hormone (ACTH)-secreting and non-functional pituitary tumors. No pathogenic germline mutations affecting the Gαi proteins were detected. The result suggests that loss-of-function mutations of GNAI loci are rare or nonexistent in familial pituitary adenomas.