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Background: Mutations in GNAS drive pancreatic tumorigenesis and frequently occur in intraductal papillary mucinous neoplasm (IPMN); however, their value as a therapeutic target is yet to be determined. This study aimed at evaluating the involvement of mutant GNAS in tumor aggressiveness in established pancreatic cancer. Methods: CRISPR/Cas9-mediated GNAS R201H silencing was performed using human primary IPMN-associated pancreatic cancer cells. The role of oncogenic GNAS in tumor maintenance was evaluated by conducting cell culture and xenograft experiments, and western blotting and transcriptome analyses were performed to uncover GNAS-driven signatures. Results: Xenografts of GNAS wild-type cells were characterized by a higher Ki-67 labeling index relative to GNAS-mutant cells. Phenotypic alterations in the GNAS wild-type tumors resulted in a significant reduction in mucin production accompanied by solid with massive stromal components. Transcriptional profiling suggested an apparent conflict of mutant GNAS with KRAS signaling. A significantly higher Notch intercellular domain (NICD) was observed in the nuclear fraction of GNAS wild-type cells. Meanwhile, inhibition of protein kinase A (PKA) induced NICD in GNAS-mutant IPMN cells, suggesting that NOTCH signaling is negatively regulated by the GNAS-PKA pathway. GNAS wild-type cells were characterized by a significant invasive property relative to GNAS-mutant cells, which was mediated through the NOTCH regulatory pathway. Conclusions: Oncogenic GNAS induces mucin production, not only via MUC2 but also via MUC5AC/B, which may enlarge cystic lesions in the pancreas. The mutation may also limit tumor aggressiveness by attenuating NOTCH signaling; therefore, such tumor-suppressing effects must be considered when therapeutically inhibiting the GNAS pathway.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) are among the most important cellular signaling components, especially G protein-coupled receptors (GPCRs). G proteins comprise three subunits, Gα, Gβ, and Gγ. Gα is the key subunit, and its structural state regulates the active status of G proteins. Interaction of guanosine diphosphate (GDP) or guanosine triphosphate (GTP) with Gα switches G protein into basal or active states, respectively. Genetic alteration in Gα could be responsible for the development of various diseases due to its critical role in cell signaling. Specifically, loss-of-function mutations of Gαs are associated with parathyroid hormone-resistant syndrome such as inactivating parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) signaling disorders (iPPSDs), whereas gain-of-function mutations of Gαs are associated with McCune–Albright syndrome and tumor development. In the present study, we analyzed the structural and functional implications of natural variants of the Gαs subtype observed in iPPSDs. Although a few tested natural variants did not alter the structure and function of Gαs, others induced drastic conformational changes in Gαs, resulting in improper folding and aggregation of the proteins. Other natural variants induced only mild conformational changes but altered the GDP/GTP exchange kinetics. Therefore, the results shed light on the relationship between natural variants of Gα and iPPSDs.

BACKGROUND: In fibrous dysplasia/McCune-Albright syndrome (FD/MAS), mosaic mutations in the GNAS gene lead to locally abnormal bone turnover. Additionally, patients with FD/MAS, particularly with thoracic lesions, have an increased risk for breast cancer. Development and progression of breast cancer has been associated with expression of Receptor Activator of NF-κB ligand (RANKL) in mammary tissue, and due to the GNAS mutation, RANKL is systemically increased in patients with FD/MAS. Yet it is unknown whether breast cancer in FD/MAS is also dependent on RANKL. We hypothesized that the GNAS mutation might induce RANKL overproduction and an oncogenic niche in mammary tissue, and examined RANKL expression in breast cancer tissue of patients with FD/MAS compared to controls.METHODS: Nine patients with FD/MAS and breast cancer were included and clinical data were retrieved. Patients were matched to controls with breast cancer without FD/MAS based on age and tumor type. Three pregnant breast cancer patients were included as positive controls. Immunohistochemical detection of RANKL was performed on formalin-fixed paraffin-embedded breast cancer specimens. Staining intensity was classified as weak, moderate or intense. The area of positive RANKL staining divided by the total ductal-lobular area was assessed (positive area percentage, PAP). Number of patients with RANKL expression was compared between FD/MAS and control group by chi-square (χ 2) test, the PAP by Mann-Whitney U test (MWU). RESULTS: RANKL expression was observed in 3 patients with FD/MAS (38 %), mainly in healthy tissue, and none of the control patients (χ 2p = 0.055). The FD/MAS group demonstrated considerably more intense staining than the control group, comparable to positive controls. The median PAP was 0.64 % (range 0.14-2.04 %) in the 3 FD/MAS patients with RANKL expression, 0.01 % (Q1-Q3: 0.0003-0.514 %) in the entire FD/MAS group, 0.006 % (Q1-Q3: 0.001-0.012 %) in the control group (MWU = 0.574), and 0.19 % (0.08-0.32 %) in the pregnant patients. All patients with FD/MAS and RANKL expression had thoracic bone lesions, but no correlation was observed between RANKL expression and presence of the GNAS mutation or FD disease burden. CONCLUSIONS: The triad of a higher number of patients, higher positive area percentage and stronger intensity in the FD/MAS compared to the control group indicates that RANKL may be upregulated in mammary tissue in a subset of patients with FD/MAS, which may explain the increased risk for breast cancer, although the clinical significance remains unclear. Further research is needed to establish risk profiles for the development of RANKL-positive breast cancer and to improve early screening and treatment.

La Heteroplasia Ósea Progresiva (HOP) es una enfermedad genética ultra rara causada por una mutación inactivantes del gen GNAS que produce formación de hueso extraesquelético a partir de tejido mesodérmico como son piel y músculo. Este trabajo presenta el único caso reportado en el mundo de HOP en dos gemelas univitelinas idénticas (edad: 11 años), que presentan la misma mutación de novo en heterocigosis en el gen GNAS pero con un desarrollo discordante de la enfermedad, mientras una de ellas permanece asintomática, la otra sufre una forma inusualmente rápida e incapacitante de la enfermedad. 2024-01-10

Background We previously reported a familial thyroid follicular cell carcinoma (FCC) in a large number of Dutch German longhaired pointers and identified two deleterious germline mutations in the TPO gene associated with disease predisposition. However, the somatic mutation profile of the FCC in dogs has not been investigated at a genome-wide scale. Results Herein, we comprehensively investigated the somatic mutations that potentially contribute to the inherited tumor formation and progression using high depth whole-genome sequencing. A GNAS p.A204D missense mutation was identified in 4 out of 7 FCC tumors by whole-genome sequencing and in 20 out of 32 dogs’ tumors by targeted sequencing. In contrast to this, in the human TC, mutations in GNAS gene have lower prevalence. Meanwhile, the homologous somatic mutation in humans has not been reported. These findings suggest a difference in the somatic mutation landscape between TC in these dogs and human TC. Moreover, tumors with the GNAS p.A204D mutation had a significantly lower somatic mutation burden in these dogs. Somatic structural variant and copy number alterations were also investigated, but no potential driver event was identified. Conclusion This study provides novel insight in the molecular mechanism of thyroid carcinoma development in dogs. German longhaired pointers carrying GNAS mutations in the tumor may be used as a disease model for the development and testing of novel therapies to kill the tumor with somatic mutations in the GNAS gene.

Chondrosarcoma (CS) is a rare tumour type and the most common primary malignant bone cancer in adults. The prognosis, currently based on tumour grade, imaging and anatomical location, is not reliable, and more objective biomarkers are required. We aimed to determine whether the level of circulating tumour DNA (ctDNA) in the blood of CS patients could be used to predict outcome. In this multi‐institutional study, we recruited 145 patients with cartilaginous tumours, of which 41 were excluded. ctDNA levels were assessed in 83 of the remaining 104 patients, whose tumours harboured a hotspot mutation in IDH1/2 or GNAS. ctDNA was detected pre‐operatively in 31/83 (37%) and in 12/31 (39%) patients postoperatively. We found that detection of ctDNA was more accurate than pathology for identification of high‐grade tumours and was associated with a poor prognosis; ctDNA was never associated with CS grade 1/atypical cartilaginous tumours (ACT) in the long bones, in neoplasms sited in the small bones of the hands and feet or in tumours measuring less than 80 mm. Although the results are promising, they are based on a small number of patients, and therefore, introduction of this blood test into clinical practice as a complementary assay to current standard‐of‐care protocols would allow the assay to be assessed more stringently and developed for a more personalised approach for the treatment of patients with CS., In this multi‐institutional study, we detected the amount of circulating tumour DNA (ctDNA) in the blood of patients with chondrosarcoma. We recruited 145 patients, and ctDNA levels were assessed in patients with tumours carrying IDH1/2 or GNAS mutations. We found that ctDNA levels offered a more accurate identification of high‐grade tumours than pathology and were additionally associated with poor prognosis.

Background McCune-Albright syndrome (MAS) is caused by postzygotic somatic mutations of the GNAS gene. It is characterized by the clinical triad of fibrous dysplasia, cafe-au-lait skin spots, and endocrinological dysfunction. Myriad complications in MAS, including hepatobiliary manifestations, are also reported. Case summary This is a case of a 4-year-old boy who presented with MAS with neonatal cholestasis. He was suspected to have Alagille syndrome due to neonatal cholestasis with intrahepatic bile duct paucity in liver biopsy, peripheral pulmonary artery stenosis, and renal tubular dysfunction. By the age of 2 years, his cholestatic liver injury gradually improved, but he had repeated left femoral fractures. He did not exhibit endocrinological abnormality or cafe-au-lait skin spots. However, MAS was suspected due to fibrous dysplasia at the age of 4 years. No mutation was identified in the GNAS gene in the DNA isolated from the peripheral blood, but an activating point mutation (c.601C>T, p.Arg201Cys) was observed in the DNA extracted from the affected bone tissue and that extracted from the formalin-fixed paraffin-embedded liver tissue, which was obtained at the age of 1 mo. Conclusion MAS should be considered as a differential diagnosis for transient cholestasis in infancy.

PTH resistance is characterized by elevated parathyroid hormone (PTH) levels, hypocalcemia, hyperphosphatemia and it is classically associated with GNAS locus genetic or epigenetic defects. Inactivating PTH/PTHrP signaling disorders (iPPSD) define overlapping phenotypes based on their molecular etiology. iPPSD1 is associated with PTH1R variants and variable phenotypes including ossification anomalies and primary failure of tooth eruption but no endocrine disorder. Here we report on a 10-month-old child born from consanguineous parents, who presented with mild neurodevelopmental delay, seizures, enlarged fontanelles, round face, and bilateral clinodactyly. Hand x-rays showed diffuse delayed bone age, osteopenia, short metacarpal bones and cone-shaped distal phalanges. A diagnosis of PTH resistance was made on the basis of severe hypocalcemia, hyperphosphatemia, elevated PTH and normal vitamin D levels on blood sample. The patient was treated with calcium carbonate and alfacalcidol leading to rapid bio-clinical improvement. Follow-up revealed multiple agenesis of primary teeth and delayed teeth eruption, as well as Arnold-Chiari type 1 malformation requiring a ventriculoperitoneal shunt placement. GNAS gene analysis showed no pathogenic variation, but a likely pathogenic homozygous substitution c.723C>G p.(Asp241Glu) in PTH1R gene was found by trio-based whole exome sequencing. We studied the deleterious impact of the variant on the protein conformation with bioinformatics tools. In conclusion, our study reports for the first time PTH resistance in a child with a biallelic PTH1R mutation, extending thereby the clinical spectrum of iPPSD1 phenotypes.

Acromegaly is caused by excessive secretion of GH and IGF-I mostly from somatotroph tumors. Various genetic and epigenetic factors are involved in the pathogenesis of somatotroph tumors. While somatic mutations of GNAS are the most prevalent cause of somatotroph tumors, germline mutations in various genes (AIP, PRKAR1A, GPR101, GNAS, MEN1, CDKN1B, SDHx, MAX) are also known as the cause of somatotroph tumors. Moreover, recent findings based on multiple perspectives of the pangenomic approach including genome, transcriptome, and methylome analyses, histological characterization, genomic instability, and possible involvement of miRNAs have gradually unveiled the whole landscape of the underlying mechanisms of somatotroph tumors. In this review, we will focus on the recent advances in genetic and epigenetic pathogenesis of somatotroph tumors.

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).

Background: Polycystic kidney disease (PKD) represents the most prevalent inherited progressive kidney disorder in humans. Due to complexity of the genetic network behind the disease, the molecular mechanisms of PKD are still poorly understood yet. Objectives: This study aimed to develop a ciliogenesis-associated gene network for PKD patients and comprehensively understand the molecular mechanisms underlying the disease. Method: The potential hub genes were selected based on the differential expression analysis from the GEO database. Meanwhile, the primary hub genes were further elucidated by both in vivo and in vitro experiments. Results: In this study, we established a comprehensive differentially expressed genes profile (including GNAS, PI4KB, UMOD, SLC7A13, and MIOX) for PKD patients compared with the control specimen. At the same time, enrichment analysis was utilized to demonstrate that the G-protein-related signaling and cilia assembling signaling pathways were closely associated with PKD development. The further investigations of the interaction between 2 genes (GNAS and PI4KB) with in vivo and in vitro analyses revealed that PI4KB functioned as a downstream factor for GNAS and spontaneously activated the phosphorylation of Akt into p-Akt for ciliogenesis in PKD formation. The PI4KB depletion mutant zebrafish model displayed a PKD phenotype as well as absence of primary cilia in the kidney. Conclusions: Collectively, our work discovered an innovative potential signaling pathway model for PKD formation, which provided a valuable insight for future study of the mechanism of this disease.

Purpose Pseudomyxoma peritonei (PMP) is a rare clinical malignancy syndrome characterized by the uncontrollable accumulation of copious mucinous ascites in the peritoneal cavity, resulting in “jelly belly”. The mechanism of tumor progression and mucin hypersecretion remains largely unknown, but GNAS mutation is a promising contributor. This review is to systemically summarize the biological background and variant features of GNAS, as well as the impacts of GNAS mutations on mucin expression, tumor cell proliferation, clinical-pathological characteristics, and prognosis of PMP. Methods NCBI PubMed database (in English) and WAN FANG DATA (in Chinese) were used for literature search. And NCBI Gene and Protein databases, Ensembl Genome Browser, COSMIC, UniProt, and RCSB PDB database were used for gene and protein review. Results GNAS encodes guanine nucleotide-binding protein α subunit (Gsα). The mutation sites of GNAS mutation in PMP are relatively stable, usually at Chr20: 57,484,420 (base pair: C-G) and Chr20: 57,484,421 (base pair: G-C). Typical GNAS mutation results in the reduction of GTP enzyme activity in Gsα, causing failure to hydrolyze GTP and release phosphoric acid, and eventually the continuous binding of GTP to Gsα. The activated Gsα could thus continuously promote mucin secretion through stimulating the cAMP-PKA signaling pathway, which is a possible mechanism leading to elevated mucin secretion in PMP. Conclusion GNAS mutation is one of the most important molecular biological features in PMP, with major functions to promote mucin hypersecretion.

In the United States, CRC is the third most common type of cancer and the second leading cause of cancer-related death. Although the incidence of CRC among the Hispanic population has been declining, recently, a dramatic increase in CRC incidents among HL younger than 50 years of age has been reported. The incidence of early-onset CRC is more significant in HL population (45%) than in non-Hispanic Whites (27%) and African-Americans (15%). The reason for these racial disparities and the biology of CRC in the HL are not well understood. We performed this study to understand the biology of the disease in HL patients. We analyzed formalin-fixed paraffin-embedded tumor tissue samples from 52 HL patients with mCRC. We compared the results with individual patient clinical histories and outcomes. We identified commonly altered genes in HL patients (APC, TP53, KRAS, GNAS, and NOTCH). Importantly, mutation frequencies in the APC gene were significantly higher among HL patients. The combination of mutations in the APC, NOTCH, and KRAS genes in the same tumors was associated with a higher risk of progression after first-line of chemotherapy and overall survival. Our data support the notion that the molecular drivers of CRC might be different in HL patients.

Background Beef tenderness is a complex trait of economic importance for the beef industry. Understanding the epigenetic mechanisms underlying this trait may help improve the accuracy of breeding programs. However, little is known about epigenetic effects on Bos taurus muscle and their implications in tenderness, and no studies have been conducted in Bos indicus. Results Comparing methylation profile of Bos indicus skeletal muscle with contrasting beef tenderness at 14 days after slaughter, we identified differentially methylated cytosines and regions associated with this trait. Interestingly, muscle that became tender beef had higher levels of hypermethylation compared to the tough group. Enrichment analysis of predicted target genes suggested that differences in methylation between tender and tough beef may affect signal transduction pathways, among which G protein signaling was a key pathway. In addition, different methylation levels were found associated with expression levels of GNAS, PDE4B, EPCAM and EBF3 genes. The differentially methylated elements correlated with EBF3 and GNAS genes overlapped CpG islands and regulatory elements. GNAS, a complex imprinted gene, has a key role on G protein signaling pathways. Moreover, both G protein signaling pathway and the EBF3 gene regulate muscle homeostasis, relaxation, and muscle cell-specificity. Conclusions We present differentially methylated loci that may be of interest to decipher the epigenetic mechanisms affecting tenderness. Supported by the previous knowledge about regulatory elements and gene function, the methylation data suggests EBF3 and GNAS as potential candidate genes and G protein signaling as potential candidate pathway associated with beef tenderness via methylation.

Background Malignant transformation of fibrous dysplasia (FD) is very rare and little is known about this occurrence. Methods We present the detailed clinical course of three cases of osteosarcoma arising from FD of the jaws and explore the genetic aberrations by Sanger sequencing, whole‐exome sequencing (WES) and immunohistochemistry (IHC). A literature review of important topics related to this occurrence was also performed. Results It was observed that patients with secondary sarcoma from FD showed a wide range of ages, with most during the third decade. Female and males were equally affected. Craniofacial bones and femurs were the most affected sites. High‐risk factors for this occurrence included polyostotic FD, McCune‐Albright syndrome and excess growth hormone. Notably, a potential relationship between thyroid hormones and sarcoma development was suggested in one patient, who began to show malignant features after hypothyroidism correction. Sanger sequencing revealed GNAS mutations of FD retained in all malignant tissues. Additionally, abnormal TP53 was demonstrated in all three cases by WES and IHC. WES also revealed two other driver mutations, ROS1 and CHD8, and large amounts of somatic copy number alterations (CNAs) where various oncogenes and tumour suppressors are located. Conclusion This study demonstrated and reviewed the clinical features and risk factors for a rare occurrence, secondary sarcoma from FD, and provided important new knowledge about its genetics., Whole exome sequencing revealed the underlying genetic mechanisms of osteosarcoma arising from fibrous dysplasia of the jaws. Significant somatic single nucleotide variations, including TP53, ROS1 AND CHD8, as well as large amounts of somatic copy number alterations were demonstrated.

The Gsα/cAMP signaling pathway mediates the effect of a variety of hormones and factors that regulate the homeostasis of the post-natal skeleton. Hence, the dysregulated activity of Gsα due to gain-of-function mutations (R201C/R201H) results in severe architectural and functional derangements of the entire bone/bone marrow organ. While the consequences of gain-of-function mutations of Gsα have been extensively investigated in osteoblasts and in bone marrow osteoprogenitor cells at various differentiation stages, their effect in adipogenically-committed bone marrow stromal cells has remained unaddressed. We generated a mouse model with expression of GsαR201C driven by the Adiponectin (Adq) promoter. Adq-GsαR201C mice developed a complex combination of metaphyseal, diaphyseal and cortical bone changes. In the metaphysis, GsαR201C caused an early phase of bone resorption followed by bone deposition. Metaphyseal bone formation was sustained by cells that were traced by Adq-Cre and eventually resulted in a high trabecular bone mass phenotype. In the diaphysis, GsαR201C, in combination with estrogen, triggered the osteogenic activity of Adq-Cre-targeted perivascular bone marrow stromal cells leading to intramedullary bone formation. Finally, consistent with the previously unnoticed presence of Adq-Cre-marked pericytes in intraosseous blood vessels, GsαR201C caused the development of a lytic phenotype that affected both cortical (increased porosity) and trabecular (tunneling resorption) bone. These results provide the first evidence that the Adq-cell network in the skeleton not only regulates bone resorption but also contributes to bone formation, and that the Gsα/cAMP pathway is a major modulator of both functions.

Pseudo-anodontia consists in the clinical, not radiographic, absence of teeth, due to failure in their eruption. It has been reported as part of an extremely rare syndrome, named GAPO syndrome. Pseudo-hypoparathyroidism type 1a (PHPT-1a) is a rare condition, characterized by resistance to the parathyroid hormone (PTH), as well as to many other hormones, and resulting in hypocalcemia, hyperphosphatemia, and elevated PTH. We report here the case of a 32-year-old woman with a long-standing history of non-treated hypocalcemia, in the context of an undiagnosed PHPT-1a. She had an intellectual disability, showed clinical features of the Albright hereditary osteodystrophy (AHO) and presented signs of multiple hormone resistances. She received treatment for seizures since the age of six. Examination of her mouth revealed a complete absence of teeth. Treatment of hypocalcemia and hormone deficiencies were started only at 29 years of age. Genetic testing demonstrated the presence of a frameshift variant in the GNAS gene in the proband as well as in her mother. A Single Nucleotide Polymorphism (SNP) array analysis failed to demonstrate pathogenic copy number variants (CNVs) but showed several regions with loss of heterozygosity (LOHs) for a final percentage of 1.75%, compatible with a fifth degree of relationship. Clinical exome sequencing (CES) ruled out any damaging variants in all the teeth agenesis-related genes. In conclusion, although we performed an extensive genetic analysis in search of possible additional gene alterations that could explain the presence of the peculiar phenotypic characteristics observed in our patient, we could not find any additional genetic defects. Our results suggest that the association of genetically confirmed PHPT-1a and complete pseudo-anodontia associated with persistent patchy alopecia areata is a new additional nonclassical feature related to the GNAS pathogenic variant.

Mazabraud's syndrome (MZB) is a rare condition in which fibrous dysplasia of bone/the McCune-Albright syndrome (FD/MAS) co-exists with intramuscular myxomas. Both FD and the myxomas harbor the GNAS-mutation. Recent studies have shown that extraskeletal, GNAS-related features are associated with a more severe phenotype of FD/MAS. However, patients with MZB are often only seen by orthopedic surgeons. We therefore evaluated MZB patients seen in tertiary referral centers from the Netherlands (LUMC), USA (National Institutes of Health) and France (INSERM UMR 1033 (Lyos), Hopital Edouard Herriot). All FD/MAS patients known in these centers with an additional diagnosis of a myxoma were included. Demographic information and data on disease extent and extraskeletal manifestations of FD/MAS such as precocious puberty (PP) or cafe-au-lait patches (CAL) were retrieved from patient's medical records. Thirty MZB patients were included: 20 women (67%) and 10 men (33%). Patients received a diagnosis of MZB (median 42 years, range 16-19) significantly later than the diagnosis of FD/MAS (median 30 years, range 0-60), p < 0.01. Twenty-six patients were diagnosed with polyostotic disease (87%). In 97% the myxoma was located near the skeletal FD lesion. The combination of MZB and MAS was made in 13 patients in whom PP (n = 7), CAL (n = 7), GH-excess (n = 3) and hyperthyroidism (n = 3) were present. Other extraskeletal features were (multinodular) goiter (n = 2) and thyroid cysts (n = 1). Furthermore, in this cohort of patients with MZB several (pre-)malignant tumors were observed; ductal carcinoma in situ of the breast in 3 patients (10%), breast cancer in 1 patient (3.3%), intra pancreatic mucinous neoplasms in 3 patients (10%) and liver adenomas in 2 patients (6.6%). A total of 47% of patients with MZB had an additional extraskeletal feature such as an endocrinopathy. In MZB, 87% of patients suffer from polyostotic FD, 43% of patients have extraskeletal GNAS-features such as an hyperfunctioning endocrinopathy and 30% (pre-)malignant tumors. We therefore advocate that MZB patients should undergo a complete screening and long-term follow-up for extent of bone disease, but also extraskeletal GNAS features of FD/MAS.

Background A large number of novel, rare, and uncharacterized mutations in Tumors including thyroid cancer (TC) have been discovered. In this study, we reported two GNAS mutations in Chinese TC patients, and assessed the functional impact. Methods Two hundred and twenty five formalin‐fixed, paraffin‐embedded (FFPE) tissue specimens from Chinese TC patients were tested by NGS. The functional impact of GNAS mutations was assessed using sorting intolerant from tolerant tools (SIFT), polymorphism phenotyping v2 (POLYPHEN‐2) and the mutation assessor. The GNAS protein structure was modeled by Iterative Threading ASSEmbly Refinement (I‐TASSER), and modeling of mutations was performed and visualised by PyMOL. Results Two somatic missense GNAS gene mutations including a rare c.956A>G(p.D319G) and a novel c.1399G>T(p.A467S) were detected in 225 TC tissues (2/225, 0.89%). The novel GNAS mutation c.1399G>T(p.A467S) was predicted to benign and low impact on the protein. SIFT and Mutation Assessor predicted c.956A>G(p.D319G) will be neutral and have low impact, while Polyphen‐2 indicated the mutation may be probably damaging. c.1399G>T(p.A467S) and c.956A>G(p.D319G) molecular modeling data showed two mutations not changed in Gsα, and showed little predicted effect on protein structure. Conclusions A rare somatic GNAS gene mutations c.956A>G(p.D319G) and a novel somatic GNAS mutation c.1399G>T(p.A467S) were found in the present study, which appeared to be associated with low impact on the protein.

Albright hereditary osteodystrophy (AHO) is caused by heterozygous inactivation of GNAS, a complex locus that encodes the alpha-stimulatory subunit of GPCRs (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 GPCRs requiring Gsα (eg., PTH, TSH, 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 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 by the targeted disruption of exon 1 of Gnas differentially affects bone remodeling based on the parental inheritance of the mutation. Mice with paternally-inherited (GnasE1+/−p) and maternally-inherited (GnasE1+/−m) mutations displayed reductions in osteoblasts along the bone surface compared to wildtype. GnasE1+/−p mice displayed reduced cortical and trabecular bone parameters due to impaired bone formation and excessive bone resorption. GnasE1+/−m mice however displayed enhanced bone parameters due to increased osteoblast activity and normal bone resorption. These distinctions in bone remodeling between GnasE1+/−p and GnasE1+/−m mice appear to be secondary to changes in the bone microenvironment driven by calcitonin-resistance within GnasE1+/−m osteoclasts and therefore warrant further studies into understanding how Gsα influences osteoblast-osteoclast coupling interactions.

Background Syndromic short stature is a genetic and phenotypic heterogeneous disorder with multiple causes. This study aims to identify genetic causes in patients with syndromic short stature of unknown cause and evaluate the efficacy of the growth hormone response. Methods Trio-whole-exome sequencing was applied to identify pathogenic gene mutations in seven patents with short stature, multiple malformations, and/or intellectual disability. Whole-genome low-coverage sequencing was also performed to identify copy number variants in three patients with concurrent intellectual disability. Recombinant human growth hormone was administered to improve height in patients with an identified cause of syndromic short stature. Results Of the seven patients, three pathogenic/likely pathogenic gene mutations, including one FGFR3 mutation (c.1620C>A p.N540K), one novel GNAS mutation (c.2288C>T p.A763V), and one novel TRPS1 mutation (c.2527_c.2528dupTA p.S843fsX72), were identified in three patients. No copy number variants were identified in the three patients with concurrent intellectual disability. The proband with an FGFR3 mutation, a female 4 and 3/12 years of age, was diagnosed with hypochondroplasia. Long-acting growth hormone improved her height from 85.8 cm [− 5.05 standard deviation (SD)] to 100.4 cm (− 4.02 SD), and her increased height SD score (SDS) was 1.03 after 25 months of treatment. The proband with a GNAS mutation, a female 12 and 9/12 years of age, was diagnosed with pseudohypoparathyroidism Ia. After 14 months of treatment with short-acting growth hormone, her height improved from 139.3 cm (− 2.69 SD) to 145.0 cm (− 2.36 SD), and her increased height SDS was 0.33. Conclusions Trio-whole-exome sequencing was an important approach to confirm genetic disorders in patients with syndromic short stature of unknown etiology. Short-term growth hormone was effective in improving height in patients with hypochondroplasia and pseudohypoparathyroidism Ia.

Purpose of review This review is timely given the 2018 publication of the first international Consensus Statement for the diagnosis and management of pseudohypoparathyroidism (PHP) and related disorders. The purpose of this review is to provide the knowledge needed to recognize and manage PHP1A, pseudopseudohypoparathyroidism (PPHP) and PHP1B – the most common of the subtypes – with an overview of the entire spectrum and to provide a concise summary of management for clinical use. This review will draw from recent literature as well as personal experience in evaluating hundreds of children and adults with PHP. Recent findings Progress is continually being made in understanding the mechanisms underlying the PHP spectrum. Every year, through clinical and laboratory studies, the phenotypes are elucidated in more detail, as are clinical issues such as short stature, brachydactyly, subcutaneous ossifications, cognitive/behavioural impairments, obesity and metabolic disturbances. Headed by a European PHP consortium, experts worldwide published the first international Consensus that provides detailed guidance in a systematic manner and will lead to exponential progress in understanding and managing these disorders. Summary As more knowledge is gained from clinical and laboratory investigations, the mechanisms underlying the abnormalities associated with PHP are being uncovered as are improvements in management.

Background: Forty percent of somatotroph tumors harbor recurrent activating GNAS mutations, historically called the gsp oncogene. In gsp-negative somatotroph tumors, GNAS expression itself is highly variable, those with GNAS overexpression most resemble phenotypically those carrying the gsp oncogene. GNAS is monoallelically expressed in the normal pituitary due to methylation-based imprinting. We hypothesize that changes in GNAS imprinting of gsp-negative tumors affect GNAS expression levels and tumorigenesis. Methods: We characterized the GNAS locus in two independent somatotroph tumor cohorts: one of 23 tumors previously published (PMID: 31883967) and classified by pan-genomic analysis, and a second with 82 tumors. Results: Multi-omics analysis of the first cohort identified a significant difference between gsp-negative and gsp-positive tumors in the methylation index at the known differentially methylated region (DMR) of the GNAS A/B transcript promoter, which was confirmed in the larger series of 82 tumors. GNAS allelic expression was analyzed using a polymorphic Fok1 cleavage site in 32 heterozygous gsp-negative tumors. GNAS expression was significantly reduced in the 14 tumors with relaxed GNAS imprinting and biallelic expression, compared to 18 tumors with monoallelic expression. Tumors with relaxed GNAS imprinting showed significantly lower SSTR2 and AIP expression levels. Conclusion: Altered A/B DMR methylation was found exclusively in gsp-negative somatotroph tumors. 43% of gsp-negative tumors showed GNAS imprinting relaxation, which correlated with lower GNAS, SSTR2 and AIP expression, indicating lower sensitivity to somatostatin analogues and potentially aggressive behavior.

Summary The exocrine pancreas, consisting of ducts and acini, is the site of origin of pancreatitis and pancreatic ductal adenocarcinoma (PDAC). Our understanding of the genesis and progression of human pancreatic diseases, including PDAC, is limited because of challenges in maintaining human acinar and ductal cells in culture. Here we report induction of human pluripotent stem cells toward pancreatic ductal and acinar organoids that recapitulate properties of the neonatal exocrine pancreas. Expression of the PDAC-associated oncogene GNASR201C induces cystic growth more effectively in ductal than acinar organoids, whereas KRASG12D is more effective in modeling cancer in vivo when expressed in acinar compared with ductal organoids. KRASG12D, but not GNASR201C, induces acinar-to-ductal metaplasia-like changes in culture and in vivo. We develop a renewable source of ductal and acinar organoids for modeling exocrine development and diseases and demonstrate lineage tropism and plasticity for oncogene action in the human pancreas., Graphical abstract, Highlights • Generation and long-term maintenance of human acinar organoids from stem cells • GNASR201C induces cystic growth more effectively in ductal than in acinar organoids • KRASG12D induces cancerous lesions more often from acinar versus ductal organoids • KRASG12D in acinar organoids induces an acinar-to-ductal metaplasia-like phenotype, Huang et al. describe conditions for generation of pancreatic ductal and acinar organoids from human stem cells. Expression of KRASG12D or GNASR201C induced cell-lineage-specific phenotypes and cell-state plasticity in culture and in vivo, identifying a renewable source of ductal and acinar organoids for modeling development and diseases of the exocrine pancreas.

Forty percent of somatotroph tumors harbor recurrent activating GNAS mutations, historically called the gsp oncogene. In gsp-negative somatotroph tumors, GNAS expression itself is highly variable; those with GNAS overexpression most resemble phenotypically those carrying the gsp oncogene. GNAS is monoallelically expressed in the normal pituitary due to methylation-based imprinting. We hypothesize that changes in GNAS imprinting of gsp-negative tumors affect GNAS expression levels and tumorigenesis. We characterized the GNAS locus in two independent somatotroph tumor cohorts: one of 23 tumors previously published (PMID: 31883967) and classified by pan-genomic analysis, and a second with 82 tumors. Multi-omics analysis of the first cohort identified a significant difference between gsp-negative and gsp-positive tumors in the methylation index at the known differentially methylated region (DMR) of the GNAS A/B transcript promoter, which was confirmed in the larger series of 82 tumors. GNAS allelic expression was analyzed using a polymorphic Fok1 cleavage site in 32 heterozygous gsp-negative tumors. GNAS expression was significantly reduced in the 14 tumors with relaxed GNAS imprinting and biallelic expression, compared to 18 tumors with monoallelic expression. Tumors with relaxed GNAS imprinting showed significantly lower SSTR2 and AIP expression levels. Altered A/B DMR methylation was found exclusively in gsp-negative somatotroph tumors. 43% of gsp-negative tumors showed GNAS imprinting relaxation, which correlated with lower GNAS, SSTR2 and AIP expression, indicating lower sensitivity to somatostatin analogues and potentially aggressive behavior.

GNAS encodes the stimulatory G protein alpha-subunit (Gsα) and its large variant XLαs. Studies have suggested that XLαs is expressed exclusively paternally. Thus, XLαs deficiency is considered to be responsible for certain findings in patients with paternal GNAS mutations, such as pseudo-pseudohypoparathyroidism, and the phenotypes associated with maternal uniparental disomy of chromosome 20, which comprises GNAS. However, a study of bone marrow stromal cells (BMSC) suggested that XLαs could be biallelically expressed. Aberrant BMSC differentiation due to constitutively activating GNAS mutations affecting both Gsα and XLαs is the underlying pathology in fibrous dysplasia of bone. To investigate allelic XLαs expression, we employed next-generation sequencing and a polymorphism common to XLαs and Gsα, as well as A/B, another paternally expressed GNAS transcript. In mouse BMSCs, Gsα transcripts were 48.4 ± 0.3% paternal, while A/B was 99.8 ± 0.2% paternal. In contrast, XLαs expression varied among different samples, paternal contribution ranging from 43.0 to 99.9%. Sample-to-sample variation in paternal XLαs expression was also detected in bone (83.7–99.6%) and cerebellum (83.8 to 100%) but not in cultured calvarial osteoblasts (99.1 ± 0.1%). Osteoblastic differentiation of BMSCs shifted the paternal XLαs expression from 83.9 ± 1.5% at baseline to 97.2 ± 1.1%. In two human BMSC samples grown under osteoinductive conditions, XLαs expression was also predominantly monoallelic (91.3 or 99.6%). Thus, the maternal GNAS contributes significantly to XLαs expression in BMSCs but not osteoblasts. Altered XLαs activity may thus occur in certain cell types irrespective of the parental origin of a GNAS defect.

The wider use of gastrointestinal endoscopic procedures has led to an increased detection of small intestinal preneoplastic and neoplastic epithelial lesions, most of which are identified in the duodenum and ampullary region. Like their malignant counterparts, small intestinal glandular precursor lesions, which include adenomas and hamartomas, may arise sporadically or be associated with hereditary tumor syndromes, such as familial adenomatous polyposis, MUTYH-associated polyposis, Lynch syndrome, Peutz-Jeghers syndrome, juvenile polyposis syndrome, and Cowden syndrome. In addition, dysplastic, preinvasive lesions have been observed adjacent to small bowel adenocarcinomas complicating immune-related disorders, such as celiac or Crohn’s disease. Adenomatous lesions may exhibit an intestinal-type, gastric-type, or, very rarely, serrated differentiation, related to different molecular pathogenetic mechanisms. Finally, in the background of multiple endocrine neoplasia 1 syndrome, precursor neuroendocrine growths have been described. In this review we offer a comprehensive description on the histo-molecular features of the main histotypes of small bowel epithelial precursors lesions, including: (i) sporadic adenomas (intestinal-type and gastric-type; non-ampullary and ampullary); (ii) syndromic adenomas; (iii) small bowel dysplasia in celiac and Crohn’s disease; (iv) serrated lesions; (v) hamartomatous lesions; and (vi) neuroendocrine precursor lesions.

Ritodrine, a β2-adrenergic receptor agonist, is among most commonly prescribed tocolytic agents. This study aimed to evaluate the associations of single nucleotide polymorphisms in GNAS, RGS2, and RGS5 with the risk of ritodrine-induced adverse events (AEs) and develop a risk scoring system to identify high-risk patients. This is the prospective cohort study conducted at the Ewha Woman’s University Mokdong Hospital between January 2010 and October 2016. Pregnant women were included if they were treated with ritodrine for preterm labor with regular uterine contractions (at least 3 every 10 min) and cervical dilation. A total of 6, 3, and 5 single nucleotide polymorphisms (SNPs) of GNAS, RGS2, and RGS5 genes were genotyped and compared in patients with and without ritodrine-induced AEs. A total of 163 patients were included in this study. After adjusting confounders, GNAS rs3730168 (per-allele odds ratio (OR): 2.1; 95% confidence interval (95% CI): 1.0–4.3) and RGS2 rs1152746 (per-allele OR: 2.6, 95% CI: 1.1–6.5) were significantly associated with ritodrine-induced AEs. According to the constructed risk scoring models, patients with 0, 1, 2, 3, 4, and 5 points showed 0%, 13%, 19%, 31%, 46%, and 100% risks of AEs. This study suggested that GNAS and RGS2 polymorphisms could affect the risk of AEs in patients treated with ritodrine.

Epigenetic alterations at imprinted genes on different chromosomes have been linked to several imprinting disorders (IDs) such as Beckwith-Wiedemann syndrome (BWS) and pseudohypoparathyroidism type 1b (PHP1b). Here, we present a male patient with these two distinct IDs caused by two independent mechanisms-loss of methylation (LOM) at chromosome 11p15.5 associated with multi-locus imprinting disturbances (MLID and paternal uniparental disomy of chromosome 20 (patUPD20). A clinical diagnosis of BWS was made based on the clinical features of macrosomia, macroglossia, and umbilical hernia. The diagnosis of PHP1b was supported by the presence of reduced growth velocity and mild learning disability as well as hypocalcemia and hyperphosphatemia at 14 years of age. Molecular analyses, including genome-wide DNA methylation (Illumina 450k array), bisulfite pyrosequencing, single nucleotide polymorphism (SNP) array and microsatellite analysis, demonstrated loss of methylation (LOM) at IC2 on chromosome 11p15.5, and paternal isodisomy of the entire chromosome 20. In addition, imprinting disturbances were noted at the differentially methylated regions (DMRs) associated with DIRAS3 on chromosome 1 and PLAGL1 on chromosome 6. This is the first case report of PHP1b due to patUPD20 diagnosed in a BWS patient with LOM at IC2 demonstrating etiologic heterogeneity for multiple imprinting disorders in a single individual.

Personalized invitro models for dysplasia and carcinogenesis in the pancreas have been constrained by insufficient differentiation of human pluripotent stem cells (hPSCs) into the exocrine pancreatic lineage. Here, we differentiate hPSCs into pancreatic duct-like organoids (PDLOs) with morphological, transcriptional, proteomic, and functional characteristics of human pancreatic ducts, further maturing upon transplantation into mice. PDLOs are generated from hPSCs inducibly expressing oncogenic GNAS, KRAS, or KRAS with genetic covariance of lost CDKN2A and from induced hPSCs derived from a McCune-Albright patient. Each oncogene causes a specific growth, structural, and molecular phenotype invitro. While transplanted PDLOs with oncogenic KRAS alone form heterogenous dysplastic lesions or cancer, KRAS with CDKN2A loss develop dedifferentiated pancreatic ductal adenocarcinomas. In contrast, transplanted PDLOs with mutant GNAS lead to intraductal papillary mucinous neoplasia-like structures. Conclusively, PDLOs enable invitro and invivo studies of pancreatic plasticity, dysplasia, and cancer formation from a genetically defined background.

Context Pseudohypoparathyroidism (PHP) is a group of disorders characterized by hypocalcemia, hyperphosphatemia, and elevated parathyroid hormone (PTH) levels as a result of end-organ resistance to PTH. Objective To describe a cohort of 26 patients with PHP followed in a single tertiary center. Methods Clinical, biochemical, radiological, and genetic analysis of the GNAS gene in 26 patients recruited since 2002. Results Ten patients harbored a GNAS mutation, 15 epigenetic abnormalities at the GNAS locus, and 1 did not show genetic or epigenetic abnormalities. According to clinical, biochemical, and genetic features, patients were classified as PHP1A, PHP1B, and pseudopseudohypoparathyroidism. Patients with PHP1A had an earlier diagnosis and more cases with family history, Albright hereditary osteodystrophy (AHO) features, hormonal resistance, and hypertension. Obesity was a common feature. No difference in biochemical values was present among PHP1A and PHP1B. Intracerebral calcification occurred in 72% of patients with no difference among PHP1A and PHP1B subgroups. No significant difference was observed between patients with and without intracerebral calcification for the time-weighted average values of total serum calcium, phosphate, calcium–phosphate product, and PTH fold increase. A borderline association between cerebral calcification and age at the time of diagnosis (P = .04) was found in the whole cohort of patients. No renal calcifications were found in the overall cohort. Conclusion Patients with PHP1A more frequently have AHO features as well as hypertension than patients with PHP1B. Patients with PHP presented a high rate of intracerebral calcification with no significant difference between subgroups. No increased risk of renal calcifications was also found in the entire cohort.

Heterotopic ossification (HO), the formation of bone outside of the skeleton, occurs in response to severe trauma and in rare genetic diseases such as progressive osseous heteroplasia (POH). In POH, which is caused by inactivation of GNAS, a gene that encodes the alpha stimulatory subunit of G proteins (Gsα), HO typically initiates within subcutaneous soft tissues before progressing to deeper connective tissues. To mimic POH, we used conditional Gnas-null mice which form HO in subcutaneous tissues upon Gnas inactivation. In response to Gnas inactivation, we determined that prior to detection of heterotopic bone, dermal adipose tissue changed dramatically, with progressively decreased adipose tissue volume and increased density of extracellular matrix over time. Upon depletion of the adipose tissue, heterotopic bone progressively formed in those locations. To investigate the potential relevance of the tissue microenvironment for HO formation, we implanted Gnas-null or control mesenchymal progenitor cells into Gnas-null or control host subcutaneous tissues. We found that mutant cells in a Gnas-null tissue environment induced a robust HO response while little/no HO was detected in control hosts. Additionally, a Gnas-null tissue environment appeared to support the recruitment of control cells to heterotopic bone, although control cell implants were associated with less HO formation compared to mutant cells. Our data support that Gnas inactivation alters the tissue microenvironment to influence mutant and wild-type progenitor cells to contribute to HO formation.

Summary Using unbiased kinase profiling, we identified protein kinase A (PKA) as an active kinase in small cell lung cancer (SCLC). Inhibition of PKA activity genetically, or pharmacologically by activation of the PP2A phosphatase, suppresses SCLC expansion in culture and in vivo. Conversely, GNAS (G-protein α subunit), a PKA activator that is genetically activated in a small subset of human SCLC, promotes SCLC development. Phosphoproteomic analyses identified many PKA substrates and mechanisms of action. In particular, PKA activity is required for the propagation of SCLC stem cells in transplantation studies. Broad proteomic analysis of recalcitrant cancers has the potential to uncover targetable signaling networks, such as the GNAS/PKA/PP2A axis in SCLC.

Introduction: Pseudohypoparathyroidism (PHP) indicates a group of rare disorders characterized by end-organ resistance to various hormones, primarily parathyroid hormone (PTH). One of its most common type is PHP-Ia, which is caused by maternally inherited inactivating mutations in GNAS. In this report, we present a Chinese girl with typical features of PHP-Ia and a novel mutation of the GNAS gene. Patient concerns: A 9-year-old Chinese girl presented with recurrent epileptic seizure. Diagnosis: Biochemical and imaging findings were consistent with PHP-Ia, including typical Albright hereditary osteodystrophy phenotype (short stature, round face, brachydactyly, and mild mental retardation), PTH resistance (hypocalcemia, hyperphosphatemia, elevated serum PTH, and multiple intracranial calcification) and thyroid stimulating hormone resistance (elevated serum thyroid stimulating hormone). Interventions: The patient was given 1α-hydroxylated vitamin D (calcitriol, 0.5 ug/d), calcium carbonate and vitamin D3 tablets (1.5 g/d, including 600 mg calcium and 125 IU vitamin D3). DNA analysis of the GNAS gene was performed for the whole family. Outcomes: Investigation of the GNAS gene revealed a novel mutation c.313delG (p.Glu105Lysfs∗7) in the patient, as well as her mother. So the diagnosis of PHP-Ia was confirmed. Conclusion: The study further expands the spectrum of known GNAS mutations associated with PHP and lay emphasis on the genetic analysis of GNAS gene for identifying genetic abnormalities as well as making diagnosis and differentiation of various subtypes of PHP.

Context: Pseudohypoparathyroidism type Ia (PHP1A) is caused by inactivating mutations involving GNAS exons 1–13, encoding the alpha-subunit of the stimulatory G protein (Gsα). Particularly PHP1A, but also other disorders involving the Gsα-cAMP-signaling pathway, have been associated with early-onset obesity. Thus, patients with mutations in the genes encoding PDE4D and PRKAR1A can also be obese. Furthermore, epigenetic GNAS changes, as in pseudohypoparathyroidism type Ib (PHP1B), can lead to excessive weight.Objective: Search for genetic variants in GNAS, PDE4D, and PRKAR1A and for methylation alterations at the GNAS locus in Finnish subjects with isolated severe obesity before age 10 years.Methods: Next generation sequencing to identify pathogenic variants in the coding exons of GNAS, PDE4D, and PRKAR1A; Multiplex Ligation-dependent Probe Amplification (MLPA) and methylation-sensitive MLPA (MS-MLPA) to search for deletions in GNAS and STX16, and for epigenetic changes at the four differentially methylated regions (DMR) within GNAS.Results: Among the 88 subjects (median age 13.8 years, median body mass index Z-score +3.9), we identified one rare heterozygous missense variant of uncertain significance in the XL exon of GNAS in a single patient. We did not identify clearly pathogenic variants in PDE4D and PRKAR1A, and no GNAS methylation changes were detected by MS-MLPA.Conclusions: Our results suggest that coding GNAS mutations or methylation changes at the GNAS DMRs, or coding mutations in PDE4D and PRKAR1A are not common causes of isolated childhood obesity in Finland.

G protein αs (GNAS) mediates receptor-stimulated cAMP signalling, which integrates diverse environmental cues with intracellular responses. GNAS is mutationally activated in multiple tumour types, although its oncogenic mechanisms remain elusive. We explored this question in pancreatic tumourigenesis where concurrent GNAS and KRAS mutations characterize pancreatic ductal adenocarcinomas (PDAs) arising from intraductal papillary mucinous neoplasms (IPMNs). By developing genetically engineered mouse models, we show that GnasR201C cooperates with KrasG12D to promote initiation of IPMN, which progress to invasive PDA following Tp53 loss. Mutant Gnas remains critical for tumour maintenance in vivo. This is driven by protein-kinase-A-mediated suppression of salt-inducible kinases (Sik1–3), associated with induction of lipid remodelling and fatty acid oxidation. Comparison of Kras-mutant pancreatic cancer cells with and without Gnas mutations reveals striking differences in the functions of this network. Thus, we uncover Gnas-driven oncogenic mechanisms, identify Siks as potent tumour suppressors, and demonstrate unanticipated metabolic heterogeneity among Kras-mutant pancreatic neoplasms.

Background Pseudohypoparathyroidism type 1B (PHP1B; MIM#603233) is a rare imprinting disorder (ID), associated with the GNAS locus, characterized by parathyroid hormone (PTH) resistance in the absence of other endocrine or physical abnormalities. Sporadic PHP1B cases, with no known underlying primary genetic lesions, could represent true stochastic errors in early embryonic maintenance of methylation. Previous data confirmed the existence of different degrees of methylation defects associated with PHP1B and suggested the presence of mosaicism, a phenomenon already described in the context of other IDs. Results With respect to mosaic conditions, the study of multiple tissues is a necessary approach; thus, we investigated somatic cell lines (peripheral blood and buccal epithelium and cells from the urine sediment) descending from different germ layers from 19 PHP patients (11 spor-PHP1B, 4 GNAS mutated PHP1A, and 4 PHP with no GNAS (epi)genetic defects) and 5 healthy controls. We identified 11 patients with epigenetic defects, further subdivided in groups with complete or partial methylation defects. The recurrence of specific patterns of partial methylation defects limited to specific CpGs was confirmed by checking methylation profiles of spor-PHP1B patients diagnosed in our lab (n = 56). Underlying primary genetic defects, such as uniparental disomy or deletion, potentially causative for the detected partial methylation were excluded in all samples. Conclusions Our data showed no differences of methylation levels between organs and tissues from the same patient, so we concluded that the epimutation occurred in early post-zygotic phases and that the partial defects were mosaics. The number of patients with no detectable (epi)genetic GNAS defects was too small to exclude epimutations occurring in later post-zygotic phases, affecting only selected tissues different from blood, thus leading to underdiagnosis during routine molecular diagnosis. Finally, we found no correlation between methylation ratios, representing the proportion of epimutated cells, and the clinical presentation, further confirming the hypothesis of a threshold effect of the GNAS loss of imprinting leading to an “all-or-none” phenotype. Electronic supplementary material The online version of this article (10.1186/s13148-018-0449-4) contains supplementary material, which is available to authorized users.

Fibrous dysplasia may show locally aggressive behaviour reflecting secondary intralesional changes, extension to soft tissue, or malignant transformation. We report the case of a patient with polyostotic fibrous dysplasia who had a giant mandibular lesion consisting of histologically typical, genotypically-confirmed, fibrous dysplasia merged with a fibrotic and hypocellular desmoplastic fibroma-like tissue in which the same Gsα-R201H mutation was detected. The occurrence of the same mutation in both the fibrous dysplasia and areas of desmoplastic fibroma suggests that the fibroma-like tissue reflects an unusual secondary tissue change within an otherwise typical fibrous dysplasia. To the best of our knowledge, only four cases of fibrous dysplasia with desmoplastic fibroma-like tissue changes have been reported.

Fibrous dysplasia (FD) of bone is a complex disease of the skeleton caused by dominant activating mutations of the GNAS locus encoding for the α subunit of the G protein-coupled receptor complex (Gsα). The mutation involves a substitution of arginine at position 201 by histidine or cysteine (GsαR201H or R201C), which leads to overproduction of cAMP. Several signaling pathways are implicated downstream of excess cAMP in the manifestation of disease. However, the pathogenesis of FD remains largely unknown. The overall FD phenotype can be attributed to alterations of skeletal stem/progenitor cells which normally develop into osteogenic or adipogenic cells (in cis), and are also known to provide support to angiogenesis, hematopoiesis, and osteoclastogenesis (in trans). In order to dissect the molecular pathways rooted in skeletal stem/progenitor cells by FD mutations, we engineered human skeletal stem/progenitor cells with the GsαR201C mutation and performed transcriptomic analysis. Our data suggest that this FD mutation profoundly alters the properties of skeletal stem/progenitor cells by pushing them towards formation of disorganized bone with a concomitant alteration of adipogenic differentiation. In addition, the mutation creates an altered in trans environment that induces neovascularization, cytokine/chemokine changes and osteoclastogenesis. In silico comparison of our data with the signature of FD craniofacial samples highlighted common traits, such as the upregulation of ADAM (A Disintegrin and Metalloprotease) proteins and other matrix-related factors, and of PDE7B (Phosphodiesterase 7B), which can be considered as a buffering process, activated to compensate for excess cAMP. We also observed high levels of CEBPs (CCAAT-Enhancer Binding Proteins) in both data sets, factors related to browning of white fat. This is the first analysis of the reaction of human skeletal stem/progenitor cells to the introduction of the FD mutation and we believe it provides a useful background for further studies on the molecular basis of the disease and for the identification of novel potential therapeutic targets. Published version

Genomic imprinting in domestic animals contributes to the variance of performance traits. However, research remains to be done on large-scale detection of epigenetic landscape of porcine imprinted loci including the GNAS complex locus. The purpose of this study was to generate porcine parthenogenetic fetuses and comprehensively identify imprinting patterns of the GNAS locus in transcript levels. To this end, both normally fertilized and bimaternal (uniparental) parthenogenetic porcine fetuses were generated, and whole genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) were performed to construct methylome and transcriptome, respectively. Differentially methylated regions (DMRs) between the fetuses were identified through methylome analysis, and parental-origin-specific expression patterns of transcripts were examined with transcriptome. As a result, three major DMRs were identified: paternally methylated Nesp DMR, maternally methylated Nespas-Gnasxl DMR, and maternally methylated Exon1B&ndash, Exon1A DMR. Parental-origin-specific expressions of those five DMR-affected transcripts were found, including a novel imprinted transcript, Exon1B, in pigs. In conclusion, using parthenotes, parental-origin-specific imprinting patterns in the porcine GNAS locus was comprehensively identified, and our approach paves the way for the discovery of novel imprinted genes and loci in a genomic context across species.

International audience; Pseudohypoparathyroidism type 1A (PHP1A) and pseudopseudohypoparathyroidism (PPHP) are two rare autosomal dominant disorders caused by loss-of-function mutations in the imprinted Guanine Nucleotide Binding Protein, Alpha Stimulating Activity (GNAS) gene, coding Gs α. PHP1A is caused by mutations in the maternal allele and results in Albright's hereditary osteodystrophy (AHO) and hormonal resistance, mainly to the parathormone (PTH), whereas PPHP, with AHO features and no hormonal resistance, is linked to mutations in the paternal allele. This study sought to investigate parental transmission of GNAS mutations. We conducted a retrospective study in a population of 204 families with 361 patients harboring GNAS mutations. To prevent ascertainment bias toward a higher proportion of affected children due to the way in which data were collected, we excluded from transmission analysis all probands in the ascertained sibships. After bias correction, the distribution ratio of the mutated alleles was calculated from the observed genotypes of the offspring of nuclear families and was compared to the expected ratio of 50% according to Mendelian inheritance (one-sample Z-test). Sex ratio, phenotype of the transmitting parent, and transmission depending on the severity of the mutation were also analyzed. Transmission analysis was performed in 114 nuclear families and included 250 descendants. The fertility rates were similar between male and female patients. We showed an excess of transmission from mother to offspring of mutated alleles (59%, p = .022), which was greater when the mutations were severe (61.7%, p = .023). Similarly, an excess of transmission was found when the mother had a PHP1A phenotype (64.7%, p = .036). By contrast, a Mendelian distribution was observed when the mutations were paternally inherited. Higher numbers of females within the carriers, but not in noncarriers, were also observed. The mother-specific transmission ratio distortion (TRD) and the sex-ratio imbalance associated to PHP1A point to a role of Gs α in oocyte biology or embryogenesis, with implications for genetic counseling. © 2019 American Society for Bone and Mineral Research.

Genomic imprinting in domestic animals contributes to the variance of performance traits. However, research remains to be done on large-scale detection of epigenetic landscape of porcine imprinted loci including the GNAS complex locus. The purpose of this study was to generate porcine parthenogenetic fetuses and comprehensively identify imprinting patterns of the GNAS locus in transcript levels. To this end, both normally fertilized and bimaternal (uniparental) parthenogenetic porcine fetuses were generated, and whole genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) were performed to construct methylome and transcriptome, respectively. Differentially methylated regions (DMRs) between the fetuses were identified through methylome analysis, and parental-origin-specific expression patterns of transcripts were examined with transcriptome. As a result, three major DMRs were identified: paternally methylated Nesp DMR, maternally methylated Nespas-Gnasxl DMR, and maternally methylated Exon1B–Exon1A DMR. Parental-origin-specific expressions of those five DMR-affected transcripts were found, including a novel imprinted transcript, Exon1B, in pigs. In conclusion, using parthenotes, parental-origin-specific imprinting patterns in the porcine GNAS locus was comprehensively identified, and our approach paves the way for the discovery of novel imprinted genes and loci in a genomic context across species.

A 70-year-old woman with loss of appetite was referred to our hospital for further examinations. Computed tomography revealed a low density tumor in the body of the pancreas measuring 4 cm in diameter. The main pancreatic duct was dilated on both the head and caudal side of the tumor. Magnetic resonance imaging showed the mass as a low intensity area in T1-weighted and high intensity area in T2-weighted images. Endoscopic retrograde cholangiopancreatography findings indicated that the main pancreatic duct was continuous with the lumen of the tumor. A cytological examination of pancreatic juice showed a class IV tumor. A distal pancreatectomy was performed as a curative resection procedure. The findings of hematoxylin eosin staining and mucus trait led to a diagnosis of invasive cancer derived from an intraductal papillary mucinous neoplasm (IPMN). We also performed sequencing analysis to investigate GNAS and K-RAS mutations in the tumor, though neither the GNAS mutation c602G>A nor K-RAS mutation c35G>A were observed. Cases negative for a GNAS mutation can be considered to have an increased risk of invasive cancer derived from an IPMN.

Pseudohypoparathyroidism (PHP) is a rare endocrine disorder derived from the defective activation of the cAMP pathway by the parathyroid hormone secondary to GNAS molecular defects. PHP subtypes are defined by the presence/absence of specific clinical/biochemical features. PHP1A is characterized by resistance to multiple hormones with features of Albright hereditary osteodystrophy (AHO), while pseudopseudohypoparathyroidism (PPHP) is characterized by AHO in the absence of PTH resistance. Small subsets of PHP and PPHP patients without known molecular defects have been re-diagnosed as being affected by the brachydactyly-mental retardation syndrome (BDMR), also known as the AHO-like syndrome. This study aimed to analyse 24 PHP1A and 51 PPHP patients without a molecular diagnosis for the presence of BDMR-associated 2q37 deletions to improve the differential diagnosis and to identify features that might help to avoid a misdiagnosis. Molecular investigations identified 4 deletions in 4 unrelated patients. The affected patients showed a combination of the most pathognomonic AHO features. Of note, 3 of the patients also displayed mild PTH resistance, and none of the patients developed ectopic ossifications. Our work confirmed the rarity of the misdiagnosis of BDMR in PHP patients through the identification of 4 patients bearing a 2q37 deletion in a cohort of 73 PHP patients (5.3%). Three patients with the deletion presented a PHP1A phenotype in the absence of any BDMR-specific findings. Further studies on larger case series are needed to elucidate the overlap between these clinical entities and to allow the early identification of patients.

Fibrous dysplasia is a benign, mass-forming disease of bone composed of abnormal fibrous and osseous elements that can be accompanied by endocrine dysfunction, skin pigmentation, and intramuscular myxomas. It is usually encountered as a solitary lesion in the tibia or femur but can develop in any bone and can be unifocal or multifocal. Difficulty arises when a solitary lesion is identified in an uncommon site or when there are prominent secondary changes, such as aneurysmal bone cyst (ABC). Molecular studies are available as an adjunct to histomorphology to aid distinction from other entities. GNAS mutations, present in greater than 70% of fibrous dysplasia cases, help in the distinction from primary ABC and low-grade osteosarcoma, which exhibit different molecular abnormalities. We report a case of monostotic fibrous dysplasia in a lumbar vertebral body with secondary change consisting of the solid variant of ABC.

Introduction Pseudohypoparathyroidism type 1A (PHP1A) and pseudopseudohypoparathyroidism (PPHP) (Inactivating PTH/PTHrP Signaling Disorders type 2, IPPSD2) are two rare autosomal disorders caused by loss-of-function mutations on either maternal or paternal allele, respectively, in the imprinted GNAS gene, which encodes the α subunit of the ubiquitously-expressed stimulatory G protein (Gαs). Case presentation We investigated a synonymous GNAS variant NM_001077488.2: c.108C>A / p.(Val36=) identified in a family presenting with IPPSD2 phenotype. In silico splicing prediction algorithms were in favor of a deleterious effect of this variant, by creating a new donor splicing site. The GNAS expression studies in blood suggested haploinsufficiency and showed an alternate splice product demonstrating the unmasking of a cryptic site, leading to a 34 base pairs deletion and the creation of a probable unstable RNA. We present the first familial case of IPPSD2 caused by a pathogenic synonymous variant in GNAS gene.

Background Increased urinary free cortisol in response to the oral administration of dexamethasone is a paradoxical reaction mainly reported in patients with primary pigmented nodular adrenocortical disease. Here, we describe the first case of subclinical Cushing’s syndrome represented by autonomous cortisol secretion and paradoxical response to oral dexamethasone administration, harboring an activating mutation in the α subunit of the stimulatory G protein (GNAS). Case presentation A 65-year-old woman was diagnosed with subclinical Cushing’s syndrome during an evaluation for bilateral adrenal masses. Tumors of unknown origin were found in the heart, brain, thyroid gland, colon, pancreas, and both adrenal glands. Adenocarcinoma of the sigmoid colon and systemic brown-patchy skin pigmentation were also present. Her urinary cortisol levels increased in response to oral dexamethasone, while serum dehydroepiandrosterone-sulfate was not suppressed. After right adrenalectomy, genetic analysis of the resected tumor revealed the somatic GNAS activating mutation, p.R201H. Paradoxical urinary cortisol response persisted even after unilateral adrenal resection, although serum and urinary cortisol levels were attenuated. Conclusions This patient harbored a GNAS activating mutation, and presented with a mild cortisol- and androgen-producing adrenal adenoma. Administration of oral dexamethasone paradoxically increased cortisol levels, possibly via the stimulation of the cyclic adenosine monophosphate-dependent protein kinase A signaling pathway, which is seen in patients with pigmented nodular adrenocortical disease or Carney complex. GNAS mutations may provide clues to the mechanisms of hyper-function and tumorigenesis in the adrenal cortex, especially in bilateral adrenal masses accompanied by multiple systemic tumors. Examining GNAS mutations could help physicians detect extra-adrenal malignancies, which may contribute to an improved prognosis for patients with this type of Cushing’s syndrome.

Somatic gain‐of‐function mutations of GNAS cause a spectrum of clinical phenotypes, ranging from McCune‐Albright syndrome (MAS) to isolated disease of bone, endocrine glands, and more rarely, other organs. In MAS, a syndrome classically characterized by polyostotic fibrous dysplasia (FD), café‐au‐lait (CAL) skin spots, and precocious puberty, the heterogenity of organ involvement, age of onset, and clinical severity of the disease are thought to reflect the variable size and the random distribution of the mutated cell clone arising from the postzygotic mutation. We report a case of neonatal MAS with hypercortisolism and cholestatic hepatobiliary dysfunction in which bone changes indirectly emanating from the disease genotype, and distinct from FD, led to a fatal outcome. Pulmonary embolism of marrow and bone fragments secondary to rib fractures was the immediate cause of death. Ribs, and all other skeletal segments, were free of changes of typical FD and fractures appeared to be the result of a mild‐to‐moderate degree of osteopenia. The mutated allele was abundant in the adrenal glands and liver, but not in skin, muscle, and fractured ribs, where it could only be demonstrated using a much more sensitive PNA hybridization probe‐based FRET (Förster resonance energy transfer) technique. Histologically, bilateral adrenal hyperplasia and cholestatic disease matched the abundant disease genotype in the adrenals and liver. Based on this case and other sporadic reports, it appears that gain‐of‐function mutations of GNAS underlie a unique syndromic profile in neonates characterized by CAL skin spots, hypercortisolism, hyperthyroidism, hepatic and cardiac dysfunction, and an absence (or latency) of FD, often with a lethal outcome. Taken together, our and previous cases highlight the phenotypic severity and the diagnostic and therapeutic challenges of MAS in neonates. Furthermore, our case specifically points out how secondary bone changes, unrelated to the direct impact of the mutation, may contribute to the unfavorable outcome of very early‐onset MAS. © 2018 The Authors JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.