Guanine Nucleotide Binding Protein, Alpha Stimulating Research Articles

First Reported Case of Gabriele-de Vries Syndrome with Spinal Dysraphism

Gabriele-de Vries syndrome is a rare autosomal dominant genetic disease caused by de novo pathogenic variants in the Yin Yang 1 (YY1) gene. Individuals with this syndrome present with multiple congenital anomalies, as well as a delay in development and intellectual disability. Herein, we report the case of a newborn male patient with a novel de novo pathogenic variant in the Guanine Nucleotide-Binding Protein, Alpha Stimulating (GNAS) gene, which was identified by whole-exome sequencing. Our patient suffered from a large open spinal dysraphism which was treated surgically immediately after birth. During the follow-up, facial dysmorphism, bladder and bowel incontinence, and mildly delayed motor and speech development were observed. Congenital central nervous system disorders were also confirmed radiologically. In this case report, we present our diagnostic and treatment approaches to this patient. To our knowledge, this is the first reported case of Gabriele-de Vries syndrome presenting with spinal dysraphism. Extensive genetic evaluation is the cornerstone in treatment of patients with suspected Gabriele-de Vries syndrome. However, in cases with potentially life-threatening conditions, surgery should be strongly considered.

Clinical features of Chilean patients with Fibrous Dysplasia/McCune-Albright Syndrome

Fibrous Dysplasia/McCune-Albright Syndrome (FD/MAS) is characterized by a spectrum of manifestations that may include fibrous dysplasia of bone and multiple endocrinopathies. To describe the clinical spectrum, the study and follow-up of patients with FD/MAS cared at our institution. Review of medical records of 12 pediatric and adult patients (11 women) who met the clinical and genetic diagnostic criteria for FD/ MAS. The patients' mean age at diagnosis was 4.9 ± 5.5 years. The most common initial clinical manifestation was peripheral precocious puberty (PPP) in 67% of patients and 75% had café-au-lait spots. Fibrous dysplasia was present in 75% of patients and the mean age at diagnosis was 7.9 ± 4.7 years. Ten patients had a bone scintigraphy, with an age at the first examination that varied between 2 and 38 years of age. The most frequent location of dysplasia was craniofacial and appendicular. No patient had a recorded history of cholestasis, hepatitis, or pancreatitis. In four patients, a genetic study was performed that was positive for the pathogenic variant of guanine nucleotide binding protein, alpha stimulating (GNAS). These patients demonstrate the variable nature of the clinical presentation and study of FD/MAS. It is essential to increase the index of diagnostic suspicion and adherence to international recommendations.

Mucinous Histology Is Associated with Resistance to Anti-EGFR Therapy in Patients with Left-Sided RAS/BRAF Wild-Type Metastatic Colorectal Cancer.

BackgroundLimited studies have suggested that mucinous histology is associated an attenuated response to anti-epidermal growth factor receptor (EGFR) therapy.MethodsWe conducted a single-institution, retrospective study to review the anti-EGFR response and the molecular profile of patients with left-sided microsatellite stable RAS/BRAF wild-type mucinous metastatic colorectal cancer.ResultsIn comparison to nonmucinous population (n = 98), mucinous histology (n = 20) was associated with a younger age (48 vs 54, P = .02), wild-type APC (80% vs 15.3%, P < .0001), and wild-type TP53 (40% vs 8.2%, P = .001). Guanine nucleotide binding protein, alpha stimulating (GNAS) mutations were exclusively found in mucinous tumors (20% vs 0, P < .0001). Genomic alterations associated with resistance to anti-EGFR therapy, such as ERBB2 amplification, PIK3CA mutation, MAP2K1 mutation, and KRAS amplification, were identified in patients with left-sided RAS/BRAF wild-type mucinous metastatic colorectal cancer. Mucinous histology was not associated with a worse outcome than non-mucinous histology (34.3 vs 42.2 months, P = .85). However, patients with left-sided RAS/BARF wild-type mucinous colorectal cancer treated with first-line anti-EGFR therapy had significantly worse progression-free survival (4 vs 6.5 months, hazard ratio [HR] = 5.3, 95% confidence interval [CI] 1.3-21.7, P = .01) than patients treated with the first-line vascular endothelial growth factor A antibody, bevacizumab. Anti-EGFR therapy was associated with limited responses and a short PFS across all lines of therapy in 12 patients with left-sided RAS/BRAF wild-type mucinous colorectal cancer.ConclusionsMucinous histology is associated with diminished benefits from anti-EGFR therapy in patients with left-sided RAS/BRAF wild-type colorectal cancer. These patients should be considered for bevacizumab-based therapy in the first- and second-line settings.

Methylation changes at the GNAS imprinted locus in pancreatic cystic neoplasms are important for the diagnosis of malignant cysts.

BACKGROUNDGuanine nucleotide-binding protein, alpha stimulating (GNAS) mutations are characteristic of intraductal papillary mucinous neoplasms (IPMNs). Pancreatic ductal adenocarcinomas (PDACs) harboring GNAS mutations originate in IPMNs. GNAS is a complex imprinted locus that produces five transcripts regulated by differential methylated regions, NESP55, GNASAS, GNASXL, GNAS1A, and GNAS.AIMTo evaluate if methylation changes in the differential methylated regions of GNAS locus contributed to malignant progression of pancreatic cysts.METHODSGNAS locus methylation was analyzed in archival pancreatic cyst fluid (PCF) obtained by endoscopic ultrasound with fine-needle aspiration by methylation specific–multiplex ligation dependent probe amplification. Results were normalized and analyzed using Coffalyser.Net software.RESULTSFifty-two PCF samples obtained by endoscopic ultrasound with fine-needle aspiration and previously characterized for KRAS and GNAS mutations were studied. The final diagnoses were surgical (11) and clinicopathological (41), including 30 benign cysts, 14 pre-malignant cyst, and eight malignant cysts. Methylation changes at NESP55, GNASAS, GNAS1A, and especially GNASXL were more frequent in malignant cysts, and NESP55 and GNASAS were useful for diagnosis. A combined variable defined as “GNAS locus methylation changes” was significantly associated with malignancy (6/8 malignant cysts and only 2/20 benign cysts) and improved classification. Hypermethylation in both maternally (NESP55) and paternally (GNASXL) derived promoters was found in 3/3 PDACs.CONCLUSIONThis is the first study to identify methylation changes in the GNAS locus, improving the diagnosis of malignant pancreatic cysts and suggesting a role in progression to PDAC.

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.

Overexpression of Guanine Nucleotide Binding Protein, Alpha Stimulating (GNAS) Regulates Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in High Glucose Environment by Regulating ERK/P38 Signaling Pathway

Bone marrow mesenchymal stem cells (BMSCs) can treat osteoporosis. Whether GNAS affects BMSCs osteogenic differentiation under high glucose condition is unknown. Rat BMSCs were isolated and randomly divided into control group, high glucose group and GNAS group. The BMSCs were transfected with GNAS plasmid in high glucose environment followed by analysis of GNAS expression by Real time PCR and Western blot, BMSCs proliferation by MTT assay, Caspase 3 activity, ALP activity, formation of calcified nodules by alizarin red staining, OC and BMP-2 expression by Real time PCR and expression of ERK/P38 signaling pathway protein by Western blot. In high glucose environment, GNAS expression was significantly decreased, cell proliferation was inhibited, Caspase 3 activity was increased, along with decreased ALP activity, calcified nodules formation and expression of OC, BMP-2, p-ERK1/2 and p-P38 (P &lt; 0.05). GNAS plasmid transfected into high glucose environment BMSCs can significantly promote GNAS expression and cell proliferation, decrease Caspase 3 activity, increase p-ERK1/2 and p-P38 expression, ALP activity and calcified nodules formation as well as increase OC and BMP-2 expression (P &lt; 0.05). GNAS1 expression is decreased in BMSCs cells in a high glucose environment. Overexpression of GNAS1 can inhibit the apoptosis of BMSCs by regulating the ERK/P38 signaling pathway, promote its proliferation and differentiation into osteogenic direction.

Abstract 037: Monogenic Hypertension Associated With a Pathogenic STX16 Mutation

Hypertension is the most important modifiable risk factor for death worldwide. Finding novel model mechanisms for blood-pressure (BP) regulation is an important goal and Mendelian syndromes have been very helpful in that regard. Pseudohypoparathyroidism type 1B (PHP1B) is an example in which affected individuals are invariably hypertensive for unknown reasons. We encountered a 48 year-old woman with severe, five-drug-resistant, hypertension. Her father and two aunts were also severely hypertensive. Her two young sons have developed drug-dependent hypertension before age 20 years. We measured 24-h ambulatory blood pressure and excluded all known secondary causes. However, the proband’s sons and their grandfather have the PHP1B phenotype, while our index patient and her two hypertensive aunts do not. The blood pressure phenotype in this pedigree suggested autosomal dominant inheritance. The Syntaxin 16 (STX16) gene encodes a snare protein and mutations cause PHP1B. Mutated STX16 causes methylation defects of Guanine Nucleotide Binding Protein, Alpha Stimulating (GNAS), a G protein regulator. Since GNAS is an imprinted gene, only inheritance from the mother causes the PHP1B phenotype. Subsequently, we sequenced STX16 in our kindred. We found a heterozygous deletion involving exons 5 and 6. We were also able to show complete loss of methylation in GNAS exon 1A. All hypertensive persons in our kindred have the STX16 mutation and are hypertensive. However, only those with maternal inheritance show the PHP1B phenotype. The methylation target region influenced by STX16 encompasses 7 genes. Our candidate is endothelin-3 encoded by EDN3 . Our data separate the hypertension-PHP1B phenotypes. We imply separate mechanisms are involved. We suggest new targets of blood pressure-raising relevance.

Overexpressed miR-196a accelerates osteogenic differentiation in osteoporotic mice via GNAS-dependent Hedgehog signaling pathway.

Osteoporosis (OP), a common metabolic bone disease, is accompanied by reduced bone mass, bone mineral density (BMD), as well as microstructure destruction of bone. Previously, microRNA-196a-2 (miR-196a-2) and miR-196a-3p were reported for its involvement in BMD. Herein, this study set out to identify the functional relevance of miR-196a in osteogenic differentiation in osteoporotic mice and explore the associated mechanism by establishing an OP mouse model. Guanine nucleotide binding protein, alpha stimulating (GNAS) was verified as a target gene of miR-196a, which was decreased in OP mice. Furthermore, the bone marrow stromal cells (BMSCs) were then extracted from OP mice and treated with miR-196 mimic/inhibitor or small interfering RNA against GNAS to investigate miR-196a interaction with GNAS and the Hedgehog signaling pathway. BMSCs in OP mice transfected with miR-196a mimic or si-GNAS displayed the elevated expression of Smo, ALP, Runx2, and OPN, as well as bone gla protein and tartrate-resistant acid phosphatase, elevated ALP vitality and bone formation ability as well as reduced expression of GNAS and PTCH. Taken conjointly, overexpression of miR-196a repressed GNAS expression by activating the Hedgehog signaling pathway, thus promoting osteogenic differentiation in mice with OP.

Study of major genetic factors involved in pituitary tumorigenesis and their impact on clinical and biological characteristics of sporadic somatotropinomas and non-functioning pituitary adenomas.

Genetic and functional aberrations of guanine nucleotide-binding protein, alpha stimulating (GNAS), aryl hydrocarbon receptor interacting protein (AIP), and pituitary tumor transforming gene (PTTG) are among the most prominent events in pituitary tumorigenesis. A cohort of Brazilian patients with somatotropinomas (n=41) and non-functioning pituitary adenomas (NFPA, n=21) from a single tertiary-referral center were evaluated for GNAS and AIP mutations and gene expression of AIP and PTTG. Results were compared to the clinical and biological (Ki67 and p53 expression) characteristics of tumors and their response to therapy, if applicable. Genetic analysis revealed that 27% of somatotropinomas and 4.8% of NFPA harbored GNAS mutations (P=0.05). However, no differences were observed in clinical characteristics, tumor extension, response to somatostatin analog therapy, hormonal/surgical remission rates, Ki67 index, and p53 expression between mutated and non-mutated somatotropinomas patients. PTTG overexpression (RQ mean=10.6, min=4.39, max=11.9) and AIP underexpression (RQ mean=0.56, min=0.46-max=0.92) were found in virtually all cases without a statistically significant relationship with clinical and biological tumor features. No patients exhibited somatic or germline pathogenic AIP mutations. In conclusion, mutations in GNAS and abnormal PTTG and AIP expression had no impact on tumor features and treatment outcomes in this cohort. Our data support some previous studies and point to the need for further investigations, probably involving epigenetic and transcriptome analysis, to improve our understanding of pituitary tumor behavior.

Novel Mutation in PTHLH Related to Brachydactyly Type E2 Initially Confused with Unclassical Pseudopseudohypoparathyroidism.

BackgroundAutosomal-dominant brachydactyly type E is a congenital abnormality characterized by small hands and feet, which is a consequence of shortened metacarpals and metatarsals. We recently encountered a young gentleman exhibiting shortening of 4th and 5th fingers and toes. Initially, we suspected him having pseudopseudohypoparathyroidism (PPHP) because of normal biochemical parameters, including electrolyte, Ca, P, and parathyroid hormone (PTH) levels; however, his mother and maternal grandmother had the same conditions in their hands and feet. Furthermore, his mother showed normal biochemical parameters. To the best of our knowledge, PPHP is inherited via a mutated paternal allele, owing to the paternal imprinting of GNAS (guanine nucleotide binding protein, alpha stimulating) in the renal proximal tubule. Therefore, we decided to further analyze the genetic background in this family.MethodsWhole exome sequencing was performed using genomic DNA from the affected mother, son, and the unaffected father as a negative control.ResultsWe selected the intersection between 45,490 variants from the mother and 45,646 variants from the son and excluded 27,512 overlapping variants identified from the father. By excluding homogenous and compound heterozygous variants and removing all previously reported variants, 147 variants were identified to be shared by the mother and son. Variants that had least proximities among species were excluded and finally 23 variants remained.ConclusionAmong them, we identified a defect in parathyroid hormone like hormone (PTHLH), encoding the PTH-related protein, to be disease-causative. Herein, we report a family affected with brachydactyly type E2 caused by a novel PTHLH mutation, which was confused with PPHP with unclassical genetic penetrance.

Detection of Rare Somatic GNAS Mutation in McCune-Albright Syndrome Using a Novel Peptide Nucleic Acid Probe in a Single Tube.

McCune-Albright syndrome (MAS) is characterized by the triad of precocious puberty, café au lait pigmentation, and polyostotic fibrous dysplasia (FD) of bone, and is caused by post-zygotic somatic mutations—R201H or R201C—in the guanine nucleotide binding protein, alpha stimulating (GNAS) gene. In the present study, a novel peptide nucleic acid (PNA) probe with fluorescent labeling was designed to detect trace amounts of somatic mutant GNAS in a single tube reaction. The method was applied to screen GNAS mutations in six patients with MAS/FD. The results showed that the PNA probe assay could detect low abundant mutants in 200-fold excess of wild-type alleles. The GNAS mutation was found in three patients with severe disease (MAS) by using the assay. The other three patients with mild disease (having only FD) showed a wild-type result. This study has provided a simple method to detect trace amounts of GNAS mutants with high sensitivity in large amounts of wild-type DNA.

The GNASR201C mutation associated with clonal hematopoiesis supports transplantable hematopoietic stem cell activity

Genome sequencing efforts have identified virtually all of the important mutations in adult myeloid malignancies. More recently, population studies have identified cancer-associated variants in the blood of otherwise healthy individuals as they age, a phenomenon termed clonal hematopoiesis of indeterminate potential (CHIP). This suggests that these mutations may occur in hematopoietic stem cells (HSCs) long before any clinical presentation but are not necessarily harbingers of transformation because only a fraction of individuals with CHIP develop hematopoietic pathologies. Delineation between CHIP variants that predispose for disease versus those that are more benign could be used as a prognostic factor to identify individuals at greater risk for transformation. To achieve this, the biological impact of CHIP variants on HSC function must be validated. One variant that has been identified recurrently in CHIP is a gain-of-function missense mutation in the imprinted gene GNAS (Guanine Nucleotide Binding Protein, Alpha Stimulating). In this study, we examined the effect of the GNASR201C variant on HSC function. Ectopic expression of GNASR201C supported transplantable HSC activity and improved lymphoid output in secondary recipients. Because declining lymphoid output is a hallmark of aging, GNASR201C mutations may sustain lymphoid-biased HSCs over time and maintain them in a developmental state favorable for transformation.

Mucinous Cystadenoma in Children and Adolescents

Study ObjectiveMucinous cystadenomas (MCAs) are benign epithelial ovarian tumors that occur rarely in children and adolescents. Because children and adolescents typically have their childbearing years ahead of them, conservative therapy is indicated. However, there is concern that ovarian cystectomy might be associated with significant recurrence risk in patients with MCA. Furthermore, guanine nucleotide binding protein, alpha stimulating (GNAS) gene mutations are associated with McCune-Albright syndrome, which is associated with cystic ovaries. We sought to evaluate the outcomes of children and adolescents with MCA treated conservatively. A subset of patients underwent GNAS gene testing. Design, Setting, Participants, and InterventionsAfter institutional board review approval, the pathology database of a large urban children's hospital was queried to identify adolescents with MCA between the years 2008 and 2014. Fourteen patients, aged 8-18 years (median, 14), were identified. A buccal swab for genetic testing was obtained from a subset of consenting patients. Main Outcome MeasuresMCA recurrence; ovarian return to normal size; GNAS gene variants. ResultsTwo patients underwent oophorectomies, and the remaining 12 underwent cystectomies. Follow-up ultrasound examination revealed slow return of ovary to normal size. Of the 10 patients with available follow-up data, there were no recurrences at a median of 225 days from surgery. Four patients consented to a buccal swab for genetic testing, and the GNAS gene was noted to have rare variants in 2 patients. ConclusionThis series supports the use of ovary-sparing surgery in the treatment of MCA. Further research exploring possible genetic variants such as the GNAS gene in children and adolescents diagnosed with MCA is warranted.

Abstract P6-09-46: A comprehensive analysis of GNAS DNA copy number, levels of mRNA and protein expression in primary breast cancer

Abstract Background: Result of recent advances in genetics, guanine nucleotide binding protein, alpha stimulating (GNAS), transducer of signals from G-protein coupled receptors, has been noted that this factor is related with the onset and progression of tumor in various cancers. We aimed to analyze gene amplification, mRNA and protein expression of GNAS and their potential association with clinicopathological factors and prognosis in primary breast cancer. Methods: The cohort of this study included 432 primary invasive breast cancer patients treated with standard care at Kumamoto University Hospital between June 2000 and January 2011. We performed a comprehensive analysis of GNAS at the levels of gene copy number, mRNA and GNAS protein expression analyzed by qPCR, qRT-PCR and immunohistochemistry (IHC), respectively. In the IHC assessment of GNAS protein expression, an H-score&amp;lt;150 was observed in 191 patients (44.2%), who were defined as having a low protein expression level, and an H-score≥150 was observed in 241 patients (55.8%) who were defined as having a high protein expression level. Results: The median age at diagnosis was 60 (range 27-93). Three hundred fifteen (72.9%) of these were postmenopausal women. One hundred forty two patients (32.9%) had axillary lymph node metastasis. The median Ki67 labeling index was 23.6 (range 0.5-97.0). The subtypes were 321 ER+/HER2-, 24 ER+/HER2+, 34 ER-/HER2+ and 53 ER-/HER2-. Three hundred twenty one patients (75.9%) were treated with endocrine therapy and 146 patients (34.6%) chemotherapy. Most notably, a low levels of GNAS protein expression was observed in 191(44.2%) patients, and was positivity associated with Ki67 (P=0.028). Furthermore, univariate and multivariate analysis revealed that low GNAS protein expression was significantly related with poor relapse-free survival rate (Log-rank test; P=0.0013, OR:0.40, 05%CI:0.22-0.70) and breast cancer specific survival rate (Log-rank-test; P=0.041, OR:0.43, 95%CI:0.19-0.97). GNAS amplification and mRNA expression were not correlated with prognoses. Conclusion: Contrary to expectations, GNAS expression was positively related with favorable tumor characteristics. Expression levels of GNAS protein may be an independent prognostic factor for primary breast cancer. Citation Format: Tomiguchi M, Yamamoto Y, Yamamoto-Ibusuki M, Goto-Yamaguchi L, Fujiki Y, Sueta A, Takeshita T, Iwase H. A comprehensive analysis of GNAS DNA copy number, levels of mRNA and protein expression in primary breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-09-46.

Steroidogenic Acute Regulatory Protein Overexpression Correlates with Protein Kinase A Activation in Adrenocortical Adenoma

The association of pathological features of cortisol-producing adrenocortical adenomas (ACAs) with somatic driver mutations and their molecular classification remain unclear. In this study, we explored the association between steroidogenic acute regulatory protein (StAR) expression and the driver mutations activating cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling to identify the pathological markers of ACAs. Immunohistochemical staining for StAR and mutations in the protein kinase cAMP-activated catalytic subunit alpha (PRKACA), protein kinase cAMP-dependent type I regulatory subunit alpha (PRKAR1A) and guanine nucleotide binding protein, alpha stimulating (GNAS) genes were examined in 97 ACAs. The association of StAR expression with the clinical and mutational features of the ACAs was analyzed. ACAs with mutations in PRKACA, GNAS, and PRKAR1A showed strong immunopositive staining for StAR. The concordance between high StAR expression and mutations activating cAMP/PKA signaling in the ACAs was 99.0%. ACAs with high expression of StAR had significantly smaller tumor volume (P < 0.001) and higher urinary cortisol per tumor volume (P = 0.032) than those with low expression of StAR. Our findings suggest that immunohistochemical staining for StAR is a reliable pathological approach for the diagnosis and classification of ACAs with cAMP/PKA signaling-activating mutations.

APOA-I: A Possible Novel Biomarker for Metabolic Side Effects in First Episode Schizophrenia

The purpose of this study was to investigate the change in plasma protein expression in first episode schizophrenia after an 8-week treatment with risperidone, and to explore potential biomarkers for metabolic side effects associated with risperidone treatment. Eighty first-episode schizophrenia patientswere enrolled in the study. Fifteen of the 80 patients were randomly selected to undergo proteomic analysis. Plasma proteins were obtained before and after the 8-week risperidone treatment, and measured using two-dimensional gel electrophoresis (2-DE), Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry(MALDI-TOF/TOF) and peptide mass fingerprinting.Proteins with the highest fold changes after risperidone treatment were then measured for all 80 patients using enzyme linked immunosorbent assay (ELISA). The relationship between changes in plasma protein levels and changes in metabolic parameters after risperidone treatment was examined. In 15 randomly selected patients, approximately 1,500 protein spots were detected in each gel by 2-DE. Of those proteins, 22 spots showed significant difference in abundance after risperidone treatment (p's<0.05). After MALDI-TOF peptide mass fingerprinting, apolipoprotein A-I (APOA-I) and Guanine Nucleotide Binding Protein, Alpha Stimulating (GNAS), were found to have the highest fold changes.The content of APOA-I was significantly increased, and the content of GNAS was significantly decreased after risperidone treatment (p's<0.05). The analysis in the entire study sample showed similar findings in changes of APOA-I and GNAS after risperidone treatment. Further analysis showed significant relationships between changesin APOA-1 and changes in triglyceride, total cholesterol, and body mass index after controlling for age, gender and family history of diabetes. Similar analysis showed a trend positive relationship between changes in GNAS and changes in BMI. Using proteomic analysis, the study suggested that APOA-I might be a novel biomarkers related to metabolic side effects in first episode schizophrenia treated with risperidone.

Linear skin atrophy preceding calcinosis cutis in pseudo-pseudohypoparathyroidism

Albright hereditary osteodystrophy (AHO) is a syndrome caused by inactivating mutations in the GNAS (guanine nucleotide-binding protein, alpha-stimulating) gene. Patients with AHO have short stature, obesity, brachydactyly and subcutaneous calcifications. AHO can be associated with pseudohypoparathyroidism type IA (PHP-IA) with upregulation of parathyroid hormone, whereas in pseudo-pseudohypoparathyroidism (PPHP), an endocrinopathy is not present. We report the case of a 5-month-old male infant who presented with slowly progressive linear atrophic skin lesions. The histological findings showed evidence of dermal hypoplasia. The child's father had PHP-IA. Four months after presentation, the infant developed calcifications within the pre-existent atrophic lesions. No alterations in calcium metabolism were noted. Analysis of the GNAS gene identified a short duplication leading to a frameshift mutation. We conclude that linear atrophic skin lesions may be an early sign of imminent cutaneous calcifications in AHO.

GNAS transcripts in skeletal progenitors: evidence for random asymmetric allelic expression of Gs

Activating mutations of the Gsalpha gene, encoded by the guanine nucleotide-binding protein, alpha stimulating (GNAS) locus located on chromosome 20q13, underlie different clinical phenotypes characterized by skeletal lesions [fibrous dysplasia (FD) of bone], extraskeletal diseases (mainly endocrine hyperfunction and skin hyperpigmentation) and variable combinations thereof [the McCune-Albright syndrome (MAS)]. This clinical heterogeneity is commonly assumed to reflect the post-zygotic origin of the mutation. However, the pattern of imprinting of the Gsalpha gene in some human post-natal tissues suggests that parental-dependent epigenetic mechanisms may also play a role in the phenotypic effect of the mutated GNAS genotype. FD lesions are generated by mutated clonogenic osteoprogenitors that reside, along with their normal counterparts, in FD bone marrow stroma. We analyzed the allelic expression pattern of Gsalpha and other GNAS alternative transcripts in the progeny of normal and mutated clonogenic stromal cells isolated in vitro from a series of informative FD/MAS patients. We report here for the first time that the two Gsalpha alleles are unequally expressed in both normal and FD-mutated stromal clones. However, in contrast to imprinting, the ratio of Gsalpha allelic expression is randomly established in different clones from the same patient. This result suggests that a parental-independent modulation of Gsalpha expression occurs in clonogenic osteoprogenitor cells and, at the single cell level, may impact on the severity of an FD lesion. Furthermore, we show that normal and mutated clonogenic stromal cells express GNAS alternative transcripts other than the common Gsalpha, some of which may be relevant to the development of FD.

49 TRANSCRIPT LEVEL OF mRNA IN BOVINE CLONED OR RE-CLONED EMBRYOS

Gene expression in embryos played important roles during preimplantation development and had the potential to be used as an indicator for embryo viability. The purpose of this study was (1) to compare the developmental competence of cloned, or re-cloned embryos (Experiment 1); (2) to analyze the transcripts level of the related implantation, metabolic, and imprinting genes in IVF, cloned and re-cloned embryos (Experiment 2). The SCNT was performed according to the established system in our laboratory (Theriogenology 2006 65, 1800–1812). For producing cloned embryos, fetal fibroblasts as donor cells were used and a viable cloned calf was born. Recloned embryos derived from ear fibroblasts of the cloned calf, genetically same with donor fetal donor cells, were produced. The couplets were fused, chemically activated, and cultured in modified synthetic oviduct fluid (mSOF) for up to 7 days. The developmental competence up to blastocysts was observed under a microscope. The implantation (Bax, E-cad, If-tau, Hsp 70, Igf2r, and DNMT1), metabolic [LDHA (Lactate Dehydrogenase A), G6PD (Glucose-6 Phosphate Dehydrogenase), PGK (Phosphogycerate Kinase), Na/K ATPase, and Glut-1], and imprinting [GNAS (guanine nucleotide binding protein, alpha stimulating), UBE3a (ubiquitin protein ligase E3A), Mest (mesoderm specific transcript), SNRPN (small nuclear ribonucleoprotein polypeptide N), and Ndn (necdin)] genes were selected. The relative abundance (ratio to GAPDH mRNA) of gene transcripts in blastocysts was measured by conventional semi-quantitive RT-PCR. In Experiment 1, development competence of SCNT pre-implantation embryo was not different between cloned or re-cloned embryos (26% vs. 22%). In Experiment 2, the relative expression of Bax, Hsp70, If-tau, and Igf2r transcript was not different in IVF, cloned, and re-cloned embryos. Expression of E-cad and DNMT1 was higher in re-cloned embryos than any other group. Transcripts levels of LDHA, Na/K ATPase, and Glut-1 showed the similar relative abundance in IVF, cloned and re-cloned embryos. Expression of G6PD and PGF was increased in re-cloned and cloned embryos, respectively. Compared to IVF and cloned embryos, in re-cloned embryos, relative abundance of related imprinting genes (Ube3a, Mest, SNRPN, and Ndn) was increased. In conclusion, this study demonstrated that, whereas developmental competence of cloned or re-cloned embryos was not different, gene transcript levels were observed differently. It was suggested that alteration of gene expression in the re-cloned embryos derived from the cloned calf, genetically same with initial donor cells, might have affected the fetal development and births of re-cloned offspring.

Microarray analysis in the HSV-1 latently infected mouse trigeminal ganglion.

To review our previous studies regarding alterations in gene expression in HSV-1 latently infected mouse trigeminal ganglia (TGs) following treatment with immunosuppressants and hyperthermia. Uninfected and HSV-1 latently infected mice were treated with immunosuppressants or heat stressed (43 degrees C for 10 minutes). In the immunosuppressant study, 4 groups of animals were examined: (1) uninfected, not treated; (2) uninfected, drug-treated; (3) latently infected, not treated; and (4) latently infected, drug-treated. In the hyperthermia study, TG from 6 groups of mice were studied: (1) uninfected, not stressed; (2) uninfected, heat-stressed; killed at 6 hours after hyperthermia; (3) uninfected, heat-stressed, killed at 24 hours after hyperthermia; (4) latently infected, not stressed; (5) latently infected, heat-stressed, killed at 6 hours after hyperthermia; and (6) latently infected, heat-stressed, killed at 24 hours after hyperthermia. PolyA mRNA from the TGs of each group was reverse-transcribed, labeled with P, incubated on a gene array membrane, and analyzed by phosphorimaging. As a comparison and to confirm microarray results, semiquantitative RT-PCR for selected genes was also performed. The immunosuppressive drugs significantly increased expression of two genes--calpactin 1 light chain and guanine nucleotide-binding protein alpha stimulating activity polypeptide (GNAS)--in the ganglia of uninfected mice compared with untreated, uninfected mice. Ten genes were shown to be significantly increased in the latent TGs from mice treated with the immunosuppressants compared with latently infected untreated mice. These genes were prostaglandin E2 receptor EP4 subtype (PTGER4), insulin promoter factor 1 (IPF1), glutathione S-transferase mu2, cyclin D2, peripherin, plasma glutathione peroxidase, methyl CpG-binding protein 2, retinal S-antigen, ErbB2 protooncogene, and GNAS. Eight genes were shown to be significantly decreased in the HSV-1 latent TGs treated with the drugs compared with untreated latent mice. These genes were peripheral myelin protein 22, decorin, transcription factor AP-1, dystroglycan 1, myelin protein zero, mitogen-activated protein kinase 3, prothymosin beta4, and brain lipid-binding protein. The results obtained by semiquantitative RT-PCR results were similar to those obtained by microarray analysis. Six hours after heat stress, the genes whose expression was altered included the FK506-binding protein gene (decreased), the T-complex protein 1alpha subunit gene (increased), and the 94-kDa glucose-regulated protein gene (increased in uninfected TG, decreased in infected TG). Heat stress increased expression of the DNA excision repair protein ERCC5 gene 24 hours after the treatment. Genes previously reported to exhibit increased transcription 1 hour after heat stress did not continue to show significant transcriptional activation at 6 or 24 hours. Those genes whose expression is altered by immunosuppressive drug treatment may play an important role in ocular HSV-1 recurrence. Changes in gene expression in the prostaglandin pathway, a transcription factor, and an enzyme in the cell cycle are considered of special importance for HSV-1 reactivation by immunosuppression. Altered gene expression at 6 and 24 hours after heat stress was different from previously reported changes in gene expression 1 hour after hyperthermia in HSV-1 latently infected mice.