Gsα Expression Research Articles

FRI597 Impact Of Theophylline On BMI In Pseudohypoparathyroidism Type 1a - Results Of An Open Label Extension Trial

Abstract Disclosure: A.H. Shoemaker: None. J. Tamaroff: None. H. Jueppner: None. Background: Pseudohypoparathyroidism type 1a (PHP1A) is a rare genetic disorder characterized by early onset obesity, short stature and multi hormone resistance. The underlying causes are maternally inherited, heterozygous inactivating mutations in GNAS, which encodes the α-subunit of stimulatory G protein (Gsα). Due to tissue specific silencing of paternal Gsα expression, maternal GNAS mutations lead to impaired Gsα-protein coupled receptor (GPCR) function and multihormone resistance. The GPCR signaling cascade begins with increased cAMP which is rapidly degraded by phosphodiesterase (PDE). We hypothesized that paternal Gsα expression is reduced, but not absent, and therefore the nonselective PDE inhibitor theophylline may potentiate the cAMP/PKA-dependent cellular events down-stream of GPCRs. We hypothesized that theophylline would reduce BMI in PHP1A patients while being safe and well tolerated. Methods: Children and adults with PHP1A who completed the randomized, placebo controlled, double-blind clinical trials (RCT) NCT03029429 and NCT04551170 were eligible for enrollment in an open-label extension (OLE) study of theophylline treatment (NCT04240821). As the RCT is ongoing and treatment assignment is blinded, we compared baseline enrollment data (prior to any exposure to theophylline or placebo) with the last study visit in the OLE study. The RCTs are 1 year long and then patients in the OLE study are followed for up to 2 additional years. The primary outcome is change in BMI, expressed as % of the 95th %ile (BMI95) to account for differences in gender and age. Results: To date, 23 patients have enrolled in, and 16 patients have completed the RCTs. Two patients completed RCTs before the OLE was available. One patient was ineligible due to study drug noncompliance. One patient was Canadian and unable to participate due to COVID-19 travel restrictions. The remaining 12 completers enrolled in the OLE. One withdrew prior to OLE visit 2 due to difficulty with travel and is not included in the analysis. Patients were 17.2 ± 12.9 years old (range 9-55 years) and 10 of 11 were female. All participants were white, one patient was Hispanic. Patients were followed for an average of 30 months. Baseline BMI95 was 133.5 ± 19.9% and at the last OLE visit, BMI95 had decreased to 124.7 ± 18.6% (p= 0.06 by paired t-test). A decrease in BMI95 was seen in 73% of patients and 36% had a decrease in BMI95 >20%. Review of the 3 patients without improvement in BMI95 showed one had an increasing BMI95 during the RCT, followed by decreasing BMI95 during the OLE, and another patient had persistently sub-therapeutic theophylline levels. The most common adverse event attributed to the drug was transient nausea/vomiting. There were no OLE withdrawals due to adverse events. Conclusion: Most patients with PHP1A in a long term, OLE study of theophylline demonstrated an improvement in BMI95. Theophylline was safe and well tolerated. Presentation: Friday, June 16, 2023

FRI596 Randomized Clinical Trial Of Theophylline For Treatment Of Pseudohypoparathyroidism Type 1a

Abstract Disclosure: A.H. Shoemaker: None. J. Tamaroff: None. H. Jueppner: None. Background: Pseudohypoparathyroidism type 1A (PHP1A) is a rare, genetic disorder caused by heterozygous inactivating mutations involving the maternal GNAS allele, the gene that encodes the stimulatory G protein (Gsα). Due to tissue specific reduced paternal Gsα expression, several tissues, particularly proximal renal tubules and thyroid, show decreased Gsα-coupled receptor function. Resultant hormone resistances can be treated, but other aspects of PHP1A such as early-onset obesity and short stature are without effective treatment options. Gsα-coupled receptor signaling cascade begins with agonist-stimulated increase in cAMP, the second messenger which is rapidly degraded by phosphodiesterase (PDE). Given that PHP1A patients have reduced, but not completely absent, cAMP production, this clinical trial will test the hypothesis that the non-selective PDE inhibitor theophylline will improve Gsα-coupled receptor signaling. We describe the study design and baseline characteristics of PHP1A patients enrolled in a double-blind, randomized, placebo-controlled clinical trial of theophylline. Methods: A single center will enroll 34 patients. Inclusion criteria: age >=2 years old, diagnosis of PHP1A and obesity. Exclusion criteria include seizure disorder, uncontrolled hormone resistance, pregnancy, and other significant medical problems. Enrollment starts with the oldest age group (>=13 years) and safety/efficacy is reviewed by the DSMB after each block of 6 patients before opening to the next age block (7-12 and 2-6 years). Patients are randomized to theophylline vs. placebo using a permuted-block randomization (2:1) stratified by age and gender. Theophylline levels guide dosing with a goal level of 10-19 mcg/mL. Unblinded study staff review theophylline levels and the dosing protocol includes sham placebo dose adjustments. The primary outcome is change in BMI, expressed as % of the 95th %ile (BMI95), to account for age and gender. Secondary endpoints include changes in glucose tolerance and insulin secretion (insulinogenic index), rate of epiphyseal closure and hormone replacement doses. Results: To date we have screened 27 and enrolled 24 patients. Enrollment in the youngest age block (2-6 years) opened in December 2022. Enrolled patients range from 4 to 55 years old (mean 16.0 ± 10.4 years, 18 pediatric patients), 92% are female, 100% are white and 13% are Hispanic. All patients were obese with a mean BMI95 130.8 ± 23.8%. Three adult patients withdrew due to headaches, nausea/difficulty swallowing pills or travel difficulties. The most common adverse event related to study drug was transient nausea/vomiting. Conclusion: This randomized clinical trial designed to test safety and efficacy of theophylline for PHP1A is ongoing. Thus far the study drug and procedures have been safe and well tolerated. Presentation: Friday, June 16, 2023

Parental Origin of Gsα Inactivation Differentially Affects Bone Remodeling in a Mouse Model of Albright Hereditary Osteodystrophy.

ABSTRACTAlbright hereditary osteodystrophy (AHO) is caused by heterozygous inactivation of GNAS, a complex locus that encodes the alpha‐stimulatory subunit of heterotrimeric G proteins (Gsα) in addition to NESP55 and XLαs due to alternative first exons. AHO skeletal manifestations include brachydactyly, brachymetacarpia, compromised adult stature, and subcutaneous ossifications. AHO patients with maternally‐inherited GNAS mutations develop pseudohypoparathyroidism type 1A (PHP1A) with resistance to multiple hormones that mediate their actions through G protein‐coupled receptors (GPCRs) requiring Gsα (eg, parathyroid hormone [PTH], thyroid‐stimulating hormone [TSH], growth hormone–releasing hormone [GHRH], calcitonin) and severe obesity. Paternally‐inherited GNAS mutations cause pseudopseudohypoparathyroidism (PPHP), in which patients have AHO skeletal features but do not develop hormonal resistance or marked obesity. These differences between PHP1A and PPHP are caused by tissue‐specific reduction of paternal Gsα expression. Previous reports in mice have shown loss of Gsα causes osteopenia due to impaired osteoblast number and function and suggest that AHO patients could display evidence of reduced bone mineral density (BMD). However, we previously demonstrated PHP1A patients display normal‐increased BMD measurements without any correlation to body mass index or serum PTH. Due to these observed differences between PHP1A and PPHP, we utilized our laboratory's AHO mouse model to address whether Gsα heterozygous inactivation differentially affects bone remodeling based on the parental inheritance of the mutation. We identified fundamental distinctions in bone remodeling between mice with paternally‐inherited (GnasE1+/−p) versus maternally‐inherited (GnasE1+/−m) mutations, and these findings were observed predominantly in female mice. Specifically, GnasE1+/−p mice exhibited reduced bone parameters due to impaired bone formation and enhanced bone resorption. GnasE1+/−m mice, however, displayed enhanced bone parameters due to both increased osteoblast activity and normal bone resorption. These in vivo distinctions in bone remodeling between GnasE1+/−p and GnasE1+/−m mice could potentially be related to changes in the bone microenvironment driven by calcitonin‐resistance within GnasE1+/−m osteoclasts. Further studies are warranted to assess how Gsα influences osteoblast–osteoclast coupling. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Lack of GNAS Remethylation During Oogenesis May Be a Cause of Sporadic Pseudohypoparathyroidism Type Ib.

Pseudohypoparathyroidism type Ib (PHP1B) is characterized by hypocalcemia and hyperphosphatemia due to parathyroid hormone resistance in the proximal renal tubules. Maternal pathogenic STX16/GNAS variants leading to maternal epigenetic GNAS changes impair expression of the stimulatory G protein alpha-subunit (Gsα) thereby causing autosomal dominant PHP1B. In contrast, genetic defects responsible for sporadic PHP1B (sporPHP1B) remain mostly unknown. Determine whether PHP1B encountered after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) causes GNAS remethylation defects similar to those in sporPHP1B. Retrospective analysis. Nine among 36 sporPHP1B patients investigated since 2000, all with loss of methylation (LOM) at the 3 maternal GNAS differentially methylated regions (DMRs) and gain of methylation at the paternal NESP DMR, had been conceived through IVF or ICSI. Besides abnormal GNAS methylation, IVF/ICSI PHP1B cases revealed no additional imprinting defects. Three of these PHP1B patients have dizygotic twins, and 4 have IVF/ICSI-conceived siblings, all with normal GNAS methylation; 2 unaffected younger siblings were conceived naturally. Sporadic and IVF/ICSI-conceived PHP1B patients revealed indistinguishable epigenetic changes at all 4 GNAS DMRs, thus suggesting a similar underlying disease mechanism. Given that remethylation at the 3 maternal DMRs occurs during oogenesis, male factors are unlikely to cause LOM postfertilization. Instead, at least some of the sporPHP1B variants could be caused by a defect or defects in an oocyte-expressed gene that is required for fertility and for re-establishing maternal GNAS methylation imprints. It remains uncertain, however, whether the lack of GNAS remethylation alone and the resulting reduction in Gsα expression is sufficient to impair oocyte maturation.

Progression of PTH Resistance in Autosomal Dominant Pseudohypoparathyroidism Type Ib Due to Maternal STX16 Deletions.

Maternally inherited STX16 deletions that cause loss of methylation at GNAS exon A/B and thereby reduce Gsα expression are the most frequent cause of autosomal dominant pseudohypoparathyroidism type Ib (AD-PHP1B). Early identification of these disease-causing variants in the children of affected and unaffected female carriers would prompt treatment with calcium and calcitriol once parathyroid hormone (PTH) levels increase, thereby preventing hypocalcemia and associated complications. This study aimed to determine when PTH and calcium abnormalities develop after birth if a STX16 deletion is inherited maternally. Forty-four children of affected (n = 7) or unaffected (n = 7) females with a STX16 deletion were investigated for the presence of these variants. If a deletion was identified, measurement of PTH, calcium, phosphate, and thyrotropin (TSH) was advised. The STX16 deletion that causes AD-PHP1B was identified in 25 children. Pretreatment laboratory results were available for 19 of those cases. Elevated PTH levels were detected by 2 years of age, and these were progressively higher if laboratory testing was first performed after establishing the genetic defect later in life. Total serum calcium levels remained within normal limits until about 5 years of age. TSH levels showed no consistent rise over time. Establishing whether a STX16 deletion is inherited from a female carrier of a disease-causing variant rapidly establishes the diagnosis of AD-PHP1B. Several years before overt hypocalcemia developed, PTH levels increased, thereby establishing the onset of PTH resistance. Our findings provide diagnostic guidance and when treatment with calcium and calcitriol should be considered in order to prevent hypocalcemia and associated sequelae.

A Distinct Variant of Pseudohypoparathyroidism (PHP) First Characterized Some 41 Years Ago Is Caused by the 3-kbSTX16 Deletion.

ABSTRACTIn 1980, Farfel and colleagues (NEJM, 1980;303:237–42) provided first evidence for two distinct variants of pseudohypoparathyroidism (PHP) that present with hypocalcemia and impaired parathyroid hormone (PTH)‐stimulated urinary cAMP and phosphate excretion, either in the presence or absence of Albright's hereditary osteodystrophy (AHO). An “abnormal allele” and an “unexpressed allele” were considered as underlying defects, predictions that turned out to be correct for both forms of PHP. Patients affected by the first variant (now referred to as PHP1A) were later shown to be carriers of inactivating mutations involving the maternal GNAS exons encoding Gsα. Patients affected by the second variant (now referred to as PHP1B) were shown in the current study to carry a maternal 3‐kb STX16 deletion, the most frequent cause of autosomal dominant PHP1B, which is associated with loss of methylation at GNAS exon A/B that reduces or abolishes maternal Gsα expression. However, the distinct maternal mutations leading to either PHP1A or PHP1B are disease‐causing only because paternal Gsα expression in the proximal renal tubules is silenced, ie, “unexpressed.” Our findings resolve at the molecular level carefully conducted investigations reported some 41 years ago that had provided first clues for the existence of two distinct PHP variants. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Molecular Definition of Pseudohypoparathyroidism Variants.

Pseudohypoparathyroidism (PHP) and pseudopseudohypoparathyroidism (PPHP) are caused by mutations and/or epigenetic changes at the complex GNAS locus on chromosome 20q13.3 that undergoes parent-specific methylation changes at several differentially methylated regions (DMRs). GNAS encodes the alpha-subunit of the stimulatory G protein (Gsα) and several splice variants thereof. PHP type Ia (PHP1A) is caused by heterozygous inactivating mutations involving the maternal exons 1-13. Heterozygosity of these maternal GNAS mutations cause PTH-resistant hypocalcemia and hyperphosphatemia because paternal Gsα expression is suppressed in certain organs thus leading to little or no Gsα protein in the proximal renal tubules and other tissues. Besides biochemical abnormalities, PHP1A patients show developmental abnormalities, referred to as Albright’s hereditary osteodystrophy (AHO). Some, but not all of these AHO features are encountered also in patients affected by PPHP, who carry paternal Gsα-specific mutations and typically show no laboratory abnormalities. Autosomal dominant PHP type Ib (AD-PHP1B) is caused by heterozygous maternal deletions within GNAS or STX16, which are associated with loss of methylation at the A/B DMR alone or at all maternally methylated GNAS exons. Loss of methylation of exon A/B and the resulting biallelic expression of A/B transcript reduces Gsα expression thus leading to hormonal resistance. Epigenetic changes at all differentially methylated GNAS regions are also observed in sporadic PHP1B, which is the most frequent PHP1B variant. However, this disease variant remains unresolved at the molecular level, except for rare cases with paternal uniparental isodisomy or heterodisomy of chromosome 20q (patUPD20q).

Targeted Long-Read Sequencing Identifies a Retrotransposon Insertion as a Cause of Altered GNAS Exon A/B Methylation in a Family With Autosomal Dominant Pseudohypoparathyroidism Type 1b (PHP1B).

Pseudohypoparathyroidism type Ib (PHP1B) is characterized predominantly by resistance to parathyroid hormone (PTH) leading to hypocalcemia and hyperphosphatemia. These laboratory abnormalities are caused by maternal loss-of-methylation (LOM) at GNAS exon A/B, which reduces in cis expression of the stimulatory G protein α-subunit (Gsα). Paternal Gsα expression in proximal renal tubules is silenced through unknown mechanisms, hence LOM at exon A/B reduces further Gsα protein in this kidney portion, leading to PTH resistance. In a previously reported PHP1B family, affected members showed variable LOM at exon A/B, yet no genetic defect was found by whole-genome sequencing despite linkage to GNAS. Using targeted long-read sequencing (T-LRS), we discovered an approximately 2800-bp maternally inherited retrotransposon insertion nearly 1200 bp downstream of exon XL not found in public databases or in 13,675 DNA samples analyzed by short-read whole-genome sequencing. T-LRS data furthermore confirmed normal methylation at exons XL, AS, and NESP and showed that LOM comprising exon A/B is broader than previously thought. The retrotransposon most likely causes the observed epigenetic defect by impairing function of a maternally derived NESP transcript, consistent with findings in mice lacking full-length NESP mRNA and in PHP1B patients with deletion of exon NESP and adjacent intronic sequences. In addition to demonstrating that T-LRS is an effective strategy for identifying a small disease-causing variant that abolishes or severely reduces exon A/B methylation, our data demonstrate that this sequencing technology has major advantages for simultaneously identifying structural defects and altered methylation. © 2022 American Society for Bone and Mineral Research (ASBMR).

Familial Pseudohypoparathyroidism Type IB Associated with an SVA Retrotransposon Insertion in the GNAS Locus.

Loss of methylation (LOM) at GNAS-A/B:TSS-differentially methylated regions (DMRs) in the GNAS locus is observed in pseudohypoparathyroidism type 1B (PHP1B). Many PHP1B cases are sporadic, but autosomal dominant-PHP1B has a deletion involving NESP55 expressed from the maternal allele or STX16 located upstream of the GNAS locus on the maternal allele. We report the possible first familial PHP1B cases with retrotransposon insertion in the GNAS locus on the maternal allele. To our knowledge, they are the possible first cases with imprinting disorders caused by retrotransposon insertion. The two sibling cases experienced tetany and/or cramps from school age and had hypocalcemia and an increased serum intact parathyroid hormone (PTH) level together with overweight, round face, and normal intellectual levels. Methylation analysis for DMRs in the GNAS locus showed only LOM of the GNAS-A/B:TSS-DMR. Copy number abnormalities at STX16 and the GNAS locus were not detected by array comparative genomic hybridization. Whole-genome sequencing and Sanger sequencing revealed an approximately 1000-bp SVA retrotransposon insertion upstream of the first exon of A/B on the GNAS locus in these siblings. Whole-genome methylome analysis by Enzymatic Methyl-Seq in the siblings showed normal methylation status in the region surrounding the insertion site and mild LOM of the GNAS-A/B:TSS-DMR. We conducted transcriptome analysis using mRNA from skin fibroblasts and induced pluripotent stem cells (iPSCs) derived from the siblings and detected no aberrant NESP55 transcripts. Quantitative reverse-transcriptase PCR (qRT-PCR) analysis in skin fibroblasts showed increased A/B expression in the patients and no NESP55 expression, even in a control. qRT-PCR analysis in iPSCs showed decreased NESP55 expression with normal methylation status of the GNAS-NESP:TSS-DMR in the patients. The retrotransposon insertion in the siblings likely caused decreased NESP55 expression that could lead to increased A/B expression via LOM of the GNAS-A/B:TSS-DMR, subsequent reduced Gsα expression, and finally, PHP1B development. © 2022 American Society for Bone and Mineral Research (ASBMR).

A Novel GNAS Duplication Associated With Loss-of-Methylation Restricted to Exon A/B Causes Pseudohypoparathyroidism Type Ib (PHP1B).

Pseudohypoparathyroidism type Ib (PHP1B) is characterized by resistance to parathyroid hormone (PTH) leading to hypocalcemia and hyperphosphatemia, and in some cases resistance toward additional hormones. Patients affected by this disorder all share a loss-of-methylation (LOM) at the differentially methylated GNAS exon A/B, which reduces expression of the stimulatory G protein α-subunit (Gsα) from the maternal allele. This leads in the proximal renal tubules, where the paternal GNAS allele does not contribute much to expression of this signaling protein, to little or no Gsα expression thereby causing PTH resistance. We now describe a PHP1B patient with a de novo genomic GNAS duplication of approximately 88 kb, which is associated with LOM restricted to exon A/B alone. Multiplex ligation-dependent probe amplification (MLPA), comparative genomic hybridization (CGH), and whole-genome sequencing (WGS) established that the duplicated DNA fragment extends from GNAS exon AS1 (telomeric breakpoint) to a small region between two imperfect repeats just upstream of LOC105372695 (centromeric breakpoint). Our novel duplication is considerably shorter than previously described duplications/triplications in that portion of chromosome 20q13 and it does not affect methylation at exons AS and XL. Based on these and previous findings, it appears plausible that the identified genomic abnormality disrupts in cis the actions of a transcript that is required for establishing or maintaining exon A/B methylation. Our findings extend the molecular causes of PHP1B and provide additional insights into structural GNAS features that are required for maintaining maternal Gsα expression and for preventing PTH-resistance. © 2020 American Society for Bone and Mineral Research (ASBMR).

Osteocytes control myeloid cell proliferation and differentiation through Gsα-dependent and -independent mechanisms.

Osteocytes, the bone cells embedded in the mineralized matrix, control bone modeling, and remodeling through direct contact with adjacent cells and via paracrine and endocrine factors that affect cells in the bone marrow microenvironment or distant organs. Osteocytes express numerous G protein-coupled receptors (GPCRs) and thus mice lacking the stimulatory subunit of G-protein (Gsα) in osteocytes (Dmp1-GsαKO mice) have abnormal myelopoiesis, osteopenia, and reduced adipose tissue. We previously reported that the severe osteopenia and the changes in adipose tissue present in these mice were mediated by increased sclerostin, which suppress osteoblast functions and promote browning of white adipocytes. Inversely, the myeloproliferation was driven by granulocyte colony-stimulating factor (G-CSF) and administration of neutralizing antibodies against G-CSF only partially restored the myeloproliferation, suggesting that additional osteocyte-derived factors might be involved. We hypothesized that osteocytes secrete Gsα-dependent factor(s) which regulate the myeloid cells proliferation. To identify osteocyte-secreted proteins, we used the osteocytic cell line Ocy454 expressing or lacking Gsα expression (Ocy454-Gsαcont and Ocy454-GsαKO ) to delineate the osteocyte "secretome" and its regulation by Gsα. Here we reported that factors secreted by osteocytes increased the number of myeloid colonies and promoted macrophage proliferation. The proliferation of myeloid cells was further promoted by osteocytes lacking Gsα expression. Myeloid cells can differentiate into bone-resorbing osteoclasts, therefore, we hypothesized that osteocyte-secreted factors might also regulate osteoclastogenesis in a Gsα-dependent manner. Conditioned medium (CM) from Ocy454 (both Gsαcont and GsαKO ) significanlty increased the proliferation of bone marrow mononuclear cells (BMNC) and, at the same time, inhibited their differentiation into mature osteoclasts via a Gsα-dependent mechanism. Proteomics analysis of CM from Ocy454 Gsαcont and GsαKO cells identified neuropilin-1 (Nrp-1) and granulin (Grn) as osteocytic-secreted proteins upregulated in Ocy454-GsαKO cells compared to Ocy454-Gsαcont , whereas semaphorin3A was significantly suppressed. Treatment of Ocy454-Gsαcont cells with recombinant proteins or knockdown of Nrp-1 and Grn in Ocy454-GsαKO cells partially rescued the inhibition of osteoclasts, demonstrating that osteocytes control osteoclasts differentiation through Nrp-1 and Grn which are regulated by Gsα signaling.

Genetic and Epigenetic Defects at the GNAS Locus Lead to Distinct Patterns of Skeletal Growth but Similar Early-Onset Obesity.

Pseudohypoparathyroidism type 1A (PHP1A), pseudoPHP (PPHP), and PHP type 1B (PHP1B) are caused by maternal and paternal GNAS mutations and abnormal methylation at maternal GNAS promoter(s), respectively. Adult PHP1A patients are reportedly obese and short, whereas most PPHP patients are born small. In addition to parathyroid hormone (PTH) resistance, PHP1A and PHP1B patients may display early-onset obesity. Because early-onset and severe obesity and short stature are daily burdens for PHP1A patients, we aimed at improving knowledge on the contribution of the GNAS transcripts to fetal and postnatal growth and fat storage. Through an international collaboration, we collected growth and weight data from birth until adulthood for 306 PHP1A/PPHP and 220 PHP1B patients. PHP1A/PPHP patients were smaller at birth than healthy controls, especially PPHP (length Z-score: PHP1A -1.1 ± 1.8; PPHP -3.0 ± 1.5). Short stature is observed in 64% and 59% of adult PHP1A and PPHP patients. PHP1B patients displayed early postnatal overgrowth (height Z-score at 1 year: 2.2 ± 1.3 and 1.3 ± 1.5 in autosomal dominant and sporadic PHP1B) followed by a gradual decrease in growth velocity resulting in normal adult height (Z-score for both: -0.4 ± 1.1). Early-onset obesity characterizes GNAS alterations and is associated with significant overweight and obesity in adults (bodey mass index [BMI] Z-score: 1.4 ± 2.6, 2.1 ± 2.0, and 1.4 ± 1.9 in PPHP, PHP1A, and PHP1B, respectively), indicating that reduced Gsα expression is a contributing factor. The growth impairment in PHP1A/PPHP may be due to Gsα haploinsufficiency in the growth plates; the paternal XLαs transcript likely contributes to prenatal growth; for all disease variants, a reduced pubertal growth spurt may be due to accelerated growth plate closure. Consequently, early diagnosis and close follow-up is needed in patients with GNAS defects to screen and intervene in case of early-onset obesity and decreased growth velocity. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).

Elevated expression of Gsα in intrahepatic cholangiocarcinoma associates with poor prognosis

The stimulatory G protein a subunit (Gsα) plays important roles in diverse cell processes including tumorigenesis. Activating mutations in Gsα gene (GNAS) have been reported to be associated with poor prognosis in various human carcinomas. Furthermore, Gsα signaling is crucial in promoting liver regeneration by interacting with growth factor signaling, indicating that Gsα might play a promoting role in cancer development. However, little is known about the correlation between Gsα levels and clinicopathological parameters in intrahepatic cholangiocarcinoma (ICC). We performed immunoblotting to examine the expression levels of Gsα and Ki67 proteins in tumor tissues and the corresponding adjacent tissues. A total of 74 pair of specimens resected from 74 ICC patients were examined. The association between Gsα levels and clinicopathological findings and prognosis of the patients was evaluated. Western blotting demonstrated that the expression of Gsα was significantly higher in ICC tissues compared with that in their corresponding adjacent tissues. Gsα protein was highly expressed in about half of ICC tissues (48.6%, 36/74) while only 28.4% (21/74) of tumor adjacent tissues showed Gsα high expression (P=0.011). High Gsα expression in ICC was significantly associated with the numbers of tumor nodules (P=0.037) and lymph node metastases (P=0.010). Moreover, the level of Gsα was significantly and positively correlated with Ki67 expression (P<0.001). In addition, the recurrence-free survival rate and overall survival rate in the Gsα high group were significantly lower than those in the Gsα low group (P=0.004 and P=0.005, respectively). High Gsα expression is correlated with poor prognosis in ICC patients. Gsα might serve as a potential prognostic indicator of ICC.

Ablation of Gsα signaling in osteoclast progenitor cells adversely affects skeletal bone maintenance.

Gsα, the alpha stimulatory subunit of heterotrimeric G proteins that activates downstream signaling through the adenylyl cyclase and cAMP/PKA pathway, plays an important role in bone development and remodeling. The role of Gsα in mesenchymal stem cell (MSC) differentiation to osteoblasts has been demonstrated in several mouse models of Gsα inactivation. Previously, using mice with heterozygous germline deletion of Gsα (Gnas+/p-), we identified a novel additional role for Gsα in bone remodeling, and showed the importance of Gnas in maintaining bone quality by regulating osteoclast differentiation and function. In this study, we show that postnatal deletion of Gsα (CreERT2;Gnasfl/fl) leads to reduction in trabecular bone quality parameters and increased trabecular osteoclast numbers. Furthermore, mice with deletion of Gsα specifically in cells of the macrophage/osteoclast lineage (LysM-Cre;Gnasfl/fl) showed reduced trabecular bone quality and increased trabecular osteoclasts, but to a reduced extent compared to the CreERT2;Gnasfl/fl global knockout. This demonstrates that while Gsα has a cell autonomous role in osteclasts in regulating bone quality, Gsα expression in other cell types additionally contribute. In both of these mouse models, cortical bone was more subtly affected than trabecular bone. Our results support that Gsα is required postnatally to maintain trabecular bone quality and that Gsα function to maintain trabecular bone is regulated in part through a specific activity in osteoclasts.

Pseudohypoparathyroidism: one gene, several syndromes.

Pseudohypoparathyroidism (PHP) and pseudopseudohypoparathyroidism (PPHP) are caused by mutations and/or epigenetic changes at the complex GNAS locus on chromosome 20q13.3 that undergoes parent-specific methylation changes at several sites. GNAS encodes the alpha-subunit of the stimulatory G protein (Gsα) and several splice variants thereof. Heterozygous inactivating mutations involving the maternal GNAS exons 1-13 cause PHP type Ia (PHP1A). Because of much reduced paternal Gsα expression in certain tissues, such as the proximal renal tubules, thyroid, and pituitary, there is little or no Gsα protein in the presence of maternal GNAS mutations, thus leading to PTH-resistant hypocalcemia and hyperphosphatemia. When located on the paternal allele, the same or similar GNAS mutations are the cause of PPHP. Besides biochemical abnormalities, patients affected by PHP1A show developmental abnormalities, referred to as Albrights hereditary osteodystrophy (AHO). Some, but not all of these AHO features are encountered also in patients affected by PPHP, who typically show no laboratory abnormalities. Autosomal dominant PHP type Ib (AD-PHP1B) is caused by heterozygous maternal deletions within GNAS or STX16, which are associated with loss-of-methylation (LOM) at exon A/B alone or at all maternally methylated GNAS exons. LOM at exon A/B and the resulting biallelic expression of A/B transcripts reduces Gsα expression, thus leading to hormonal resistance. Epigenetic changes at all differentially methylated GNAS regions are also observed in sporadic PHP1B, the most frequent disease variant, which remains unresolved at the molecular level, except for rare cases with paternal uniparental isodisomy or heterodisomy of chromosome 20q (patUPD20q).

Gsα deficiency in the dorsomedial hypothalamus underlies obesity associated with Gsα mutations.

Gsα, encoded by Gnas, mediates hormone and neurotransmitter receptor-stimulated cAMP generation. Heterozygous Gsα-inactivating mutations lead to obesity in Albright hereditary osteodystrophy (AHO) patients, but only when the mutations occur on the maternal allele. This parent-of-origin effect is due to Gsα imprinting in the CNS, although the relevant CNS regions are unknown. We have now shown that mice with a Gnas gene deletion disrupting Gsα expression on the maternal allele, but not the paternal allele, in the dorsomedial nucleus of the hypothalamus (DMH) developed obesity and reduced energy expenditure without hyperphagia. Although maternal Gnas deletion impaired activation of brown adipose tissue (BAT) in mice, their responses to cold environment remained intact. Similar findings were observed in mice with DMH-specific deficiency of melanocortin MC4R receptors, which are known to activate Gsα. Our results show that Gsα imprinting in the DMH underlies the parent-of-origin metabolic phenotype that results from Gsα mutations and that DMH MC4R/Gsα signaling is important for regulation of energy expenditure and BAT activation, but not the metabolic response to cold.

Clinicopathological implications of GNAS in Ewing sarcoma.

The objective of the present study was to determine whether guanine nucleotide-binding protein α stimulating (GNAS) gene expression correlates with pathognomonic signs by analyzing the mutations, methylation status and G-protein α subunit (Gsα) expression of GNAS in Ewing sarcoma (ES). Formalin-fixed paraffin-embedded tissue samples from 77 patients with primary ES were obtained in South Korea, Argentina and Brazil, and were studied via methylation chip assay and direct sequencing of the GNAS gene and immunohistochemical analysis of Gsα. The mutation and methylation statuses of the GNAS gene were examined. Immunohistochemical results were measured with respect to proportion and staining intensity. The results revealed that GNAS genes in ES tumor samples were less methylated compared with normal controls. No mutations were detected at exons 8 or 9 of the GNAS locus complex on chromosome 20q13.3, indicating that the pathogenesis of ES was not associated with GNAS mutation. Gsα expression correlated well with the methylation status of the GNAS gene. Notably, high Gsα expression was detected more frequently in samples from living patients than from decedents, although this was not statistically significant (P=0.055). In conclusion, GNAS mutation is not associated with the pathogenesis of ES tumors. This finding may be used to differentiate ES tumors from metastatic bone lesions with morphological similarity to ES tumors. Analysis of the methylation status of the GNAS gene and immunohistochemical Gsα expression suggests that hypermethylated GNAS (low Gsα expression) in ES may be associated with unfavorable progression with a non-significant trend.

GNAS Spectrum of Disorders.

The GNAS complex locus encodes the alpha-subunit of the stimulatory G protein (Gsα), a ubiquitous signaling protein mediating the actions of many hormones, neurotransmitters, and paracrine/autocrine factors via generation of the second messenger cAMP. GNAS gives rise to other gene products, most of which exhibit exclusively monoallelic expression. In contrast, Gsα is expressed biallelically in most tissues; however, paternal Gsα expression is silenced in a small number of tissues through as-yet-poorly understood mechanisms that involve differential methylation within GNAS. Gsα-coding GNAS mutations that lead to diminished Gsα expression and/or function result in Albright's hereditary osteodystrophy (AHO) with or without hormone resistance, i.e., pseudohypoparathyroidism type-Ia/Ic and pseudo-pseudohypoparathyroidism, respectively. Microdeletions that alter GNAS methylation and, thereby, diminish Gsα expression in tissues in which the paternal Gsα allele is normally silenced also cause hormone resistance, which occurs typically in the absence of AHO, a disorder termed pseudohypoparathyroidism type-Ib. Mutations of GNAS that cause constitutive Gsα signaling are found in patients with McCune-Albright syndrome, fibrous dysplasia of bone, and different endocrine and non-endocrine tumors. Clinical features of these diseases depend significantly on the parental allelic origin of the GNAS mutation, reflecting the tissue-specific paternal Gsα silencing. In this article, we review the pathogenesis and the phenotypes of these human diseases.

Cholera toxin adjuvant promotes a balanced Th1/Th2/Th17 response independently of IL-12 and IL-17 by acting on Gsα in CD11b⁺ DCs.

Despite an extensive literature on the mechanism of action of cholera toxin (CT), we still lack critical information about how the toxin acts as an adjuvant and, especially, which dendritic cells (DCs) are the target cells. Although a T helper type 2 (Th2)-skewing effect of CT is most commonly reported, effective priming of Th17 cells as well as suppression of Th1 responses are well documented. However, the ability of CT to block interferon regulatory factor 8 (IRF8) function and interleukin (IL)-12 production in DCs, which blocks CD8α DC and Th1 cell development, is inconsistent with priming of Th1 and CD8 T cells in many other reports. This prompted us to investigate the adjuvant effect of CT in wild-type, IL-12p40-/-, Batf3-/-, and IL-17A-/- mice and in mice that selectively lack the Gsα target protein for CT adenosine diphosphate (ADP)-ribosylation in DCs. We found that CT promoted Th1 priming independently of IL-12, and whereas Th2 and also Th17 responses were augmented, the gut IgA responses did not require IL-17A. Adjuvanticity was intact in Batf3-/- mice, lacking CD8α(+) DCs, but completely lost in mice with Gsα-deficient CD11c cells. Thus, our data demonstrate that the adjuvant effect requires Gsα expression in CD11b(+) DCs, and that priming of mucosal IgA and CD4 T cells appears unbiased and is independent of IL-12 and IL-17A.

Fluoxetine Up-Regulates Bcl-xL Expression in Rat C6 Glioma Cells

ObjectiveTo analyze both differentially expressed genes and the Bcl-xL protein expression after acute and chronic treatment with fluoxetine in rat C6 glioma cells.MethodsC6 glioma cells were cultured for 24 h or 72 h after treatment with 10 µM fluoxetine, and gene expression patterns were observed using microarray and qRT-PCR. Then, cells were cultured for 6 h, 24 h, 72 h or 96 h after treatment with 10 µM fluoxetine, and the expression of Bcl-xL protein was measured using western blot.ResultsAs determined by microarray, treatment with fluoxetine for 24 h up-regulated 33 genes (including Bcl-xL and NCAM140) and down-regulated 7 genes (including cyclin G-associated kinase). Treatment with fluoxetine for 72 h up-regulated 53 genes (including Gsα and Bcl-xL) and down-regulated 77 genes (including Gαi2 and annexin V). Based on the qRT-PCR results, there was an increase in Gsα mRNA and a decrease in Gαi2 mRNA at 72 h in fluoxetine-treated cells as compared to control, a result that was consistent with microarray. We also observed an increase in Bcl-xL mRNA (both at 24 h and at 72 h) in fluoxetine-treated cells as compared to control, demonstrating a tendency to increase gradually. Bcl-xL protein expression increased as the duration of fluoxetine treatment increased.ConclusionThese results suggest that chronic treatment with fluoxetine not only initiates the cAMP pathway through inducing Gsα expression but also induces Bcl-xL expression, thus inhibiting apoptosis.