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GNAS variants were recently described in 1% of patients not known to have pseudohypoparathyroidism/inactivating PTH/PTHrP signalling disorder 2 in the UK Genetics of Obesity Study. We describe a new missense GNAS variant, c.791A > C, p.(Asp264Thr), in a family with obesity, hyperphagia and mild PTH resistance. A 6-year-old female (body mass index +4.3 SD score [SDS], height +1.9 SDS) presented with hyperphagia and obesity from age 3 years. She had subtle brachydactyly, macrocephaly, and mildly delayed development. The 12-year-old brother (height +2.1 SDS, body mass index +2.9 SDS) had hyperphagia, obesity, mildly delayed development, and autism. He had subtle brachydactyly, as did the affected mother. We assessed the functional effect of the mutant, measuring cAMP production in cells transfected with wild type and mutant GNAS after ligand stimulation. Cells with the mutant GNAS showed impaired cAMP generation through melanocortin receptor 4, GH releasing hormone receptor, and PTH receptor. These cases demonstrate the clinical heterogeneity of monogenic disease, suggesting a need to test for PHP1A in children with obesity even without classical signs of PHP1A. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) mediate a wide spectrum of physiological functions, including the development, remodeling, and repair of the skeleton. Fibrous dysplasia (FD) of the bone is characterized by fibrotic, expansile bone lesions caused by activating mutations in GNAS. There are no effective therapies for FD. We previously showed that ColI(2.3)+/Rs1+ mice, in which Gs-GPCR signaling was hyper-activated in osteoblastic cell lineages using an engineered receptor strategy, developed a fibrotic bone phenotype with trabecularization that could be reversed by normalizing Gs-GPCR signaling, suggesting that targeting the Gs-GPCR or components of the downstream signaling pathway could serve as a promising therapeutic strategy for FD. The Wnt signaling pathway has been implicated in the pathogenesis of FD-like bone, but the specific Wnts and which cells produce them remain largely unknown. Single-cell RNA sequencing on long-bone stromal cells of 9-wk-old male ColI(2.3)+/Rs1+ mice and littermate controls showed that fibroblastic stromal cells in ColI(2.3)+/Rs1+ mice were expanded. Multiple Wnt ligands were up- or downregulated in different cellular populations, including in non-osteoblastic cells. Treatment with the porcupine inhibitor LGK974, which blocks Wnt signaling broadly, induced partial resorption of the trabecular bone in the femurs of ColI(2.3)+/Rs1+ mice, but no significant changes in the craniofacial skeleton. Bone fibrosis remained evident after treatment. Notably, LGK974 caused significant bone loss in control mice. These results provide new insights into the role of Wnt and Gs-signaling in fibrosis and bone formation in a mouse model of Gs-GPCR pathway overactivation. [ABSTRACT FROM AUTHOR]

Objective: Pseudohypoparathyroidism type 1B (PHP1B) caused by methylation defects of differentially methylated regions (DMRs) on the GNAS locus can be categorized into groups according to etiologies and methylation defect patterns of the DMRs. The aim of this study was to clarify the clinical characteristics of each group. Design: Comprehensive molecular analyses consisting of methylation, copy number, and microsatellite analyses. Methods: Eighty-four patients with PHP1B were included in this study. We classified them into 5 groups, namely, autosomal dominant inheritance-PHP1B (Group 1, G1), sporadic-PHP1B (G2), and atypical-PHP1B (G3-G5), based on the methylation defect patterns in 4 DMRs on the GNAS locus and etiologies and evaluated the clinical findings in each group and compared them among the groups. Results: G2 had the youngest age and the highest serum intact parathyroid hormone levels among the 5 groups at the time of diagnosis. The most common symptoms at the time of diagnosis were tetany in G1, and seizures or loss of consciousness in G2. Albright's hereditary osteodystrophy and PHP-suggestive features were most frequently observed in the G2 proband. Nine patients had neurodevelopmental disorders (NDs) consisting of mild to borderline intellectual disability and/or developmental delay. There were no significant correlations between the average methylation ratios of 7 CpG sites in the GNAS-A/B:TSS-DMR and hormonal and biochemical findings. Conclusion: This study revealed the differences in some clinical characteristics, particularly clinical features, and ages at the time of diagnosis between G2 and other groups and detailed NDs observed in some patients with PHP1B. [ABSTRACT FROM AUTHOR]

Background: Pseudohypoparathyroidism type 1a (PHP1a) is a rare endocrine disease caused by partial defects of the α subunit of the stimulatory Guanosin triphosphate (GTP) binding protein (Gsα) resulting from maternal GNAS gene variation. The clinical manifestations are related to PTH resistance (hypocalcemia, hyperphosphatemia, and elevated serum intact PTH) in the presence or absence of multihormone resistance, and Albright’s hereditary osteodystrophy (AHO). Objectives: To summarize the molecular genetics results and clinical characteristics as well as to explore the correlations between them. Methods: Articles pertaining to PHP1a until May, 31, 2021 were reviewed and 527 patients with genetic diagnosis were included in the data analysis. The clinical characteristics and molecular genetics results of these patients were analyzed and compared to explore the correlations between them. Results: A total of 258 GNAS rare variants (RVs) were identified in 527 patients. The RVs were most commonly found in exons 1 and 7 (17.6% each), with frameshift (36.8%), and missense (31.3%) being the main types of RVs. The median age of onset was 5.0 years old. The most common clinical manifestations were elevation of PTH (86.7%) and AHO (87.5%). Thyroid stimulating hormone resistance was the most common hormone resistance (75.5%) other than PTH resistance. Patients with missense and in-frame RVs had lower incidence rates of the round face (P = .001) and subcutaneous ossifications (P < .001) than those with loss-of-function (non-sense, frameshift, splicing site variants, and large deletions) variants. Conclusions: This study revealed the correlation between loss-of-function RVs with round faces and subcutaneous ossifications in PHP 1a patients. Further exploration of genotype-phenotype correlations through more standardized and prospective studies with long-term follow-up is necessary. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

Objectives: To investigate the gene mutational profile of urachal carcinoma in correlation with its clinicopathologic features.Methods: We analyzed genetic mutations in 30 cases of urachal carcinoma by next-generation sequencing (NGS) test. Histologic slides and clinical data were reviewed.Results: The patients included 21 men and 9 women, with a mean age of 53 years (range, 24-75 years). The urachal carcinomas included mucinous (11), enteric (10), signet ring cell (8), and high-grade neuroendocrine (1) subtypes. Targeted NGS analysis demonstrated genetic mutations in all the urachal tumors (mean, 2; range, 1-4). TP53 was the most mutated gene (25), followed by KRAS (9) and GNAS (8) genes. TP53 mutations were more common in the signet ring cell subtype (7/8), and GNAS mutations were present only in the mucinous (5/11) and signet ring cell subtypes (3/8) but not in the enteric subtype (0/10). KRAS mutations were significantly associated with cancer stage IV (P = .02) and younger patient age (P = .046). Furthermore, the presence of KRAS mutations in urachal carcinoma portended a poorer overall survival (P = .006).Conclusions: Urachal carcinoma demonstrates frequent gene mutations that are associated with distinct clinicopathologic features. Gene mutation may underlie the development and progression of this aggressive disease. [ABSTRACT FROM AUTHOR]

Context: Sporadic pseudohypoparathyroidism type 1B (sporPHP1B) is an imprinting disease without a defined genetic cause, characterized by broad methylation changes in differentially methylated regions (DMRs) of the GNAS gene. Objective: This work aims to provide insights into the causative event leading to the GNAS methylation defects through comprehensive molecular genetic analyses of a pair of female monozygotic twins concordant for sporPHP1B who were conceived naturally, that is, without assisted reproductive techniques. Methods: Using the leukocyte genome of the twins and family members, we performed targeted bisulfite sequencing, methylation-sensitive restriction enzyme (MSRE)- quantitative polymerase chain reaction (qPCR), whole-genome sequencing (WGS), highdensity single-nucleotide polymorphism (SNP) array, and Sanger sequencing. Results: Methylation analyses by targeted bisulfite sequencing and MSRE-qPCR revealed almost complete losses of methylation at the GNAS AS, XL, and A/B DMRs and a gain of methylation at the NESP55 DMR in the twins, but not in other family members. Except for the GNAS locus, we did not find apparent methylation defects at other imprinted genome loci of the twins. WGS, SNP array, and Sanger sequencing did not detect the previously described genetic defects associated with familial PHP1B. Sanger sequencing also ruled out any novel genetic alterations in the entire NESP55/AS region. However, the analysis of 28 consecutive SNPs could not exclude the possibility of paternal heterodisomy in a span of 22 kb comprising exon NESP55 and AS exon 5. Conclusion: Our comprehensive analysis of a pair of monozygotic twins with sporPHP1B ruled out all previously described genetic causes. Twin concordance indicates that the causative event was an imprinting error earlier than the timing of monozygotic twinning. [ABSTRACT FROM AUTHOR]

Context: 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. Objective: This study aimed to determine when PTH and calcium abnormalities develop after birth if a STX16 deletion is inherited maternally. Methods: 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. Results: 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. Conclusion: 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. [ABSTRACT FROM AUTHOR]

Albright 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. [ABSTRACT FROM AUTHOR]

Context: Patients with pseudohypoparathyroidism type 1b (PHP1b) show disordered imprinting of the maternal GNAS allele or paternal uniparental disomy (UPD). Genetic deletions in STX16 or in upstream exons of GNAS are present in many familial but not sporadic cases.Objective: Characterization of epigenetic and genetic defects in patients with PHP1b.Design and Patients: DNA from 84 subjects, including 26 subjects with sporadic PHP1b, 27 affected subjects and 17 unaffected and/or obligate gene carriers from 12 PHP1b families, 11 healthy individuals, and 3 subjects with PHP1a was subjected to quantitative pyrosequencing of GNAS differentially methylated regions (DMRs), microarray analysis, and microsatellite haplotype analysis.Setting: Academic medical center.Main Outcome Measurements: Molecular pathology of PHP1b.Results: Healthy subjects, unaffected family members and obligate carriers of paternal PHP1b alleles, and subjects with PHP1a showed normal methylation of all DMRs. All PHP1b subjects showed loss of methylation (LOM) at the exon A/B DMR. Affected members of 9 PHP1b kindreds showed LOM only at the exon A/B DMR, which was associated with a 3-kb deletion of STX16 exons 4 through 6 in 7 families and a novel deletion of STX16 and adjacent NEPEPL1 in 1 family. A novel NESP deletion was found in 1 of 2 other families with more extensive methylation defects. One sporadic PHP1b had UPD of 20q, 2 had 3-kb STX16 deletions, and 5 had apparent epigenetic mosaicism.Conclusions: We found diverse patterns of defective methylation and identified novel or previously known mutations in 9 of 12 PHP1b families. [ABSTRACT FROM AUTHOR]

Context: Pseudohypoparathyroidism type 1B (PHP1B), also referred to as inactivating PTH/PTHrP signaling disorder (iPPSD), is characterized by proximal renal tubular resistance to parathyroid hormone (PTH) leading to hypocalcemia, hyperphosphatemia, and elevated PTH values. Autosomal dominant PHP1B (AD-PHP1B) with loss of methylation at the maternal GNAS A/B:TSS-DMR (transcription start site-differentially methylated region) alone can be caused by maternal deletions involving STX16.Objective: Characterize a previously not reported AD-PHP1B family with loss of methylation at GNAS A/B:TSS-DMR, but without evidence for a STX16 deletion on the maternal allele and assess GNAS-AS2:TSS-DMR methylation.Methods: DNA from 24 patients and 10 controls were investigated. AD-PHP1B patients without STX16 deletion from a single family (n = 5), AD-PHP1B patients with STX16 deletion (n = 9), sporPHP1B (n = 10), unaffected controls (n = 10), patUPD20 (n = 1), and matUPD20 (n = 1). Methylation and copy number analyses were performed by pyrosequencing, methylation-sensitive multiplex ligation-dependent probe amplification, and multiplex ligation-dependent probe amplification.Results: Molecular cloning of polymerase chain reaction-amplified, bisulfite-treated genomic DNA from healthy controls revealed evidence for 2 distinct GNAS-AS2:TSS-DMR subdomains, named AS2-1 and AS2-2, which showed 16.0 ± 2.3% and 31.0 ± 2.2% methylation, respectively. DNA from affected members of a previously not reported AD-PHP1B family without the known genetic defects revealed incomplete loss of methylation at GNAS A/B:TSS-DMR, normal methylation at the 3 well-established maternal and paternal DMRs, and, surprisingly, increased methylation at AS2-1 (32.9 ± 3.5%), but not at AS2-2 (30.5 ± 2.9%).Conclusion: The distinct methylation changes at the novel GNAS-AS2:TSS-DMR will help characterize further different PHP1B/iPPSD3 variants and will guide the search for underlying genetic defects, which may provide novel insights into the mechanisms underlying GNAS methylation. [ABSTRACT FROM AUTHOR]

Objectives: Mucinous adenocarcinoma arising in unresected congenital pulmonary airway malformation (CPAM) is rare. Underlying driver mutations in addition to KRAS gain-of-function mutations in this setting and the long-term outcomes of these patients are unknown.Methods: We report a case of metastatic mucinous adenocarcinoma harboring both KRAS and GNAS mutations arising in a type 1 CPAM of a 14-year-old male. A literature review was performed.Results: Next-generation sequencing revealed identical KRAS (G12V) mutations in both the CPAM and metastatic adenocarcinoma and a missense mutation in the GNAS (R201C) gene in the metastatic adenocarcinoma only. Median survival was 23 and 4 years for patients with localized (no or limited spread within the same lobe of CPAM) and distant involvement (spread to any different lobe of CPAM) of mucinous cells, respectively (95% confidence interval, 23-23 and 1.5-22 years, respectively; P = .017).Conclusions: Mucinous cell proliferation associated with type 1 CPAM has exceptionally good long-term outcomes if confined within the same lobe of CPAM. A second oncogenic mutation in the GNAS gene may be necessary for progression to malignancy and distant spread. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

Fibrous dysplasia (FD) is a rare bone disease caused by activating mutations of GNAS encoding the Gsα protein, enhancing cyclic adenosine monophosphate (cAMP) production by overstimulation of adenylyl cyclase and impairing osteoblastic differentiation. The clinical presentation ranges from asymptomatic to polyostotic forms with severe disability, explained by the mosaic distribution of the GNAS mutation. Physicians have to deal with the gap of knowledge in FD pathogenesis, the absence of prognostic markers and the lack of specific treatment. The identification of specific biomarkers for FD is an important step to improve the clinical and therapeutic approaches. An epigenetic regulation driven by microRNAs (miRNAs), known as promising biomarkers in bone disease, could be involved in FD. We have sought circulating miRNAs that are differentially expressed in FD patients compared to controls and would reflect dysregulations of osteogenesis‐related genes and bone disorder. The global miRNA profiling was performed using Next Generation Sequencing in patient serum collected from a discovery cohort of 20 patients (10 polyostotic and 10 monostotic) and 10 controls. From these, we selected 19 miRNAs for a miRNA validation phase from serum of 82 patients and 82 controls, using real‐time qPCR. Discovery screening identified 111 miRNAs differentially expressed in patient serum, after adjusting for the false discovery rate (FDR). Among the 82 patients, 55% were polyostotic, and 73% were women with a mean age of 42 years. Six miRNAs (miR‐25‐3p, miR‐93‐5p, miR‐182‐5p, miR‐324‐5p, miR‐363‐3p, and miR‐451a) were significantly overexpressed in serum, with FDR <0.05. The expression level of these six miRNAs was not associated with the FD severity. In conclusion, we identified a signature of circulating miRNAs associated with FD. These miRNAs are potential negative regulators of gene expression in bone cell progenitors, suggesting their activity in FD by interfering with osteoblastic and osteoclastic differentiation to impair bone mineralization and remodeling processes. © 2020 American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

Context: Pseudohypoparathyroidism type 1A (PHP1A) and pseudopseudohypoparathyroidism (PPHP) are caused by inactivating mutations in the exons of GNAS that encode the alpha-subunit of the stimulatory G protein (Gsα). In some cases abnormal methylation of exon A/B of GNAS, a hallmark of PHP1B, has been reported.Objective: To identify the underlying genetic basis for PHP1A/PPHP in patients in whom molecular defects were not detected by GNAS sequencing and microarray-based analysis of copy number variations.Methods: Whole genome sequencing (WGS) and pyrosequencing of differentially methylated regions (DMRs) of GNAS using genomic deoxyribonucleic acid from affected patients.Results: We identified 2 novel heterozygous GNAS deletions: a 6.4 kb deletion that includes exon 2 of GNAS in the first proband that was associated with normal methylation (57%) of exon A/B DMR, and a 1438 bp deletion in a second PHP1A patient that encompasses the promoter region and 5' untranslated region of Gsα transcripts, which was inherited from his mother with PPHP. This deletion was associated with reduced methylation (32%) of exon A/B DMR.Conclusions: WGS can detect exonic and intronic mutations, including deletions that are too small to be identified by microarray analysis, and therefore is more sensitive than other techniques for molecular analysis of PHP1A/PPHP. One of the deletions we identified led to reduced methylation of exon A/B DMR, further refining a region needed for normal imprinting of this DMR. We propose that deletion of this region can explain why some PHP1A patients have reduced of methylation of the exon A/B DMR. [ABSTRACT FROM AUTHOR]

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 Gsα. 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 Gsα in oocyte biology or embryogenesis, with implications for genetic counseling. © 2019 American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

ABSTRACT: 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). [ABSTRACT FROM AUTHOR]

ABSTRACT Pseudohypoparathyroidism type Ib (PHP1B) is characterized primarily by resistance to parathyroid hormone (PTH) and thus hypocalcemia and hyperphosphatemia, in most cases without evidence for Albright hereditary osteodystrophy (AHO). PHP1B is associated with epigenetic changes at one or several differentially-methylated regions (DMRs) within GNAS, which encodes the α-subunit of the stimulatory G protein (Gsα) and splice variants thereof. Heterozygous, maternally inherited STX16 or GNAS deletions leading to isolated loss-of-methylation (LOM) at exon A/B alone or at all maternal DMRs are the cause of autosomal dominant PHP1B (AD-PHP1B). In this study, we analyzed three affected individuals, the female proband and her two sons. All three revealed isolated LOM at GNAS exon A/B, whereas the proband's healthy maternal grandmother and uncle showed normal methylation at this locus. Haplotype analysis was consistent with linkage to the STX16/ GNAS region, yet no deletion could be identified. Whole-genome sequencing of one of the patients revealed a large heterozygous inversion (1,882,433 bp). The centromeric breakpoint of the inversion is located 7,225 bp downstream of GNAS exon XL, but its DMR showed no methylation abnormality, raising the possibility that the inversion disrupts a regulatory element required only for establishing or maintaining exon A/B methylation. Because our three patients presented phenotypes consistent with PHP1B, and not with PHP1A, the Gsα promoter is probably unaffected by the inversion. Our findings expand the spectrum of genetic mutations that lead to LOM at exon A/B alone and thus biallelic expression of the transcript derived from this alternative first GNAS exon. © 2017 American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

ABSTRACT The cyclic adenosine monophosphate (cAMP) intracellular signaling pathway mediates the physiological effects of several hormones and neurotransmitters, acting by the activation of G-protein coupled receptors (GPCRs) and several downstream intracellular effectors, including the heterotrimeric stimulatory G-protein (Gs), the cAMP-dependent protein kinase A (PKA), and cAMP-specific phosphodiesterases (PDEs). Defective G-protein-mediated signaling has been associated with an increasing number of disorders, including Albright hereditary osteodistrophy (AHO) and pseudohypoparathyroidism (PHP), a heterogeneous group of rare genetic metabolic disorders resulting from molecular defects at the GNAS locus. Moreover, mutations in PRKAR1A and PDE4D genes have been recently detected in patients with acrodysostosis (ACRDYS), showing a skeletal and endocrinological phenotype partially overlapping with AHO/PHP. Despite the high detection rate of molecular defects by currently available molecular approaches, about 30% of AHO/PHP patients still lack a molecular diagnosis, hence the need to screen patients negative for GNAS epi/genetic defects also for chromosomal regions and genes associated with diseases that undergo differential diagnosis with PHP. According to the growing knowledge on Gsα-cAMP signaling-linked disorders, we investigated our series of patients ( n = 81) with a clinical diagnosis of PHP/AHO but negative for GNAS anomalies for the presence of novel genetic variants at PRKAR1A and PDE4D genes. Our work allowed the detection of 8 novel missense variants affecting genes so far associated with ACRDYS in 9 patients. Our data further confirm the molecular and clinical overlap among these disorders. We present the data collected from a large series of patients and a brief review of the literature in order to compare our findings with already published data; to look for PRKAR1A/PDE4D mutation spectrum, recurrent mutations, and mutation hot spots; and to identify specific clinical features associated with ACRDYS that deserve surveillance during follow-up. © 2016 American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

Proximal tubular resistance to parathyroid hormone (PTH) resulting in hypocalcemia and hyperphosphatemia are preeminent abnormalities in pseudohypoparathyroidism type Ib (PHP1B), but resistance toward other hormones as well as variable features of Albright's Hereditary Osteodystrophy (AHO) can occur also. Genomic DNA from PHP1B patients shows epigenetic changes at one or multiple differentially methylated regions (DMRs) within GNAS, the gene encoding Gas and splice variants thereof. In the autosomal dominant disease variant, these methylation abnormalities are caused by deletions in STX16 or GNAS on the maternal allele. The molecular defect(s) leading to sporadic PHP1B (sporPHP1B) remains in most cases unknown and we therefore analyzed 60 sporPHP1B patients and available family members by microsatellite markers, single nucleotide polymorphisms (SNPs), multiplex ligationdependent probe amplification (MLPA), and methylation-specific MLPA (MS-MLPA). All investigated cases revealed broad GNAS methylation changes, but no evidence for inheritance of two paternal chromosome 20q alleles. Some patients with partial epigenetic modifications inDNAfrom peripheral blood cells showed more complete GNAS methylation changes when testing their immortalized lymphoblastoid cells. Analysis of siblings and children of sporPHP1B patients provided no evidence for an abnormal mineral ion regulation and no changes in GNAS methylation. Only one patient revealed, based on MLPA and microsatellite analyses, evidence for an allelic loss, which resulted in the discovery of two adjacent, maternally inherited deletions (37,597 and 1427 bp, respectively) that remove the area between GNAS antisense exons 3 and 5, including exon NESP. Our findings thus emphasize that the region comprising antisense exons 3 and 4 is required for establishing all maternal GNAS methylation imprints. The genetic defect(s) leading in sporPHP1B to epigenetic GNAS changes and thus PTH-resistance remains unknown, but it seems unlikely that this disease variant is caused by heterozygous inherited or de novo mutations involving GNAS. [ABSTRACT FROM AUTHOR]

ABSTRACT We recently reported the generation and initial characterization of the first direct model of human fibrous dysplasia (FD; OMIM #174800), obtained through the constitutive systemic expression of one of the disease-causing mutations, GsαR201C, in the mouse. To define the specific pathogenetic role(s) of individual cell types within the stromal/osteogenic system in FD, we generated mice expressing GsαR201C selectively in mature osteoblasts using the 2.3kb Col1a1 promoter. We show here that this results in a striking high bone mass phenotype but not in a mimicry of human FD. The high bone mass phenotype involves specifically a deforming excess of cortical bone and prolonged and ectopic cortical bone remodeling. Expression of genes characteristic of late stages of bone cell differentiation/maturation is profoundly altered as a result of expression of GsαR201C in osteoblasts, and expression of the Wnt inhibitor Sost is reduced. Although high bone mass is, in fact, a feature of some types/stages of FD lesions in humans, it is marrow fibrosis, localized loss of adipocytes and hematopoietic tissue, osteomalacia, and osteolytic changes that together represent the characteristic pathological profile of FD, as well as the sources of specific morbidity. None of these features are reproduced in mice with osteoblast-specific expression of GsαR201C. We further show that hematopoietic progenitor/stem cells, as well as more mature cell compartments, and adipocyte development are normal in these mice. These data demonstrate that effects of Gsα mutations underpinning FD-defining tissue changes and morbidity do not reflect the effects of the mutations on osteoblasts proper. © 2015 American Society for Bone and Mineral Research. © 2014 American Society for Bone and Mineral Research [ABSTRACT FROM AUTHOR]

ABSTRACT Hypocalcemia and hyperphosphatemia because of resistance toward parathyroid hormone (PTH) in the proximal renal tubules are the most prominent abnormalities in patients affected by pseudohypoparathyroidism type Ib (PHP-Ib). In this rare disorder, which is caused by GNAS methylation changes, resistance can occur toward other hormones, such as thyroid-stimulating hormone (TSH), that mediate their actions through G protein-coupled receptors. However, these additional laboratory abnormalities are usually not recognized until PTH-resistant hypocalcemia becomes clinically apparent. We now describe four pediatric patients, first diagnosed with subclinical or overt hypothyroidism between the ages of 0.2 and 15 years, who developed overt PTH-resistance 3 to 20 years later. Although anti-thyroperoxidase (anti-TPO) antibodies provided a plausible explanation for hypothyroidism in one of these patients, this and two other patients revealed broad epigenetic GNAS abnormalities, which included loss of methylation (LOM) at exons AS, XL, and A/B, and gain of methylation at exon NESP55; ie, findings consistent with PHP-Ib. LOM at GNAS exon A/B alone led in the fourth patient to the identification of a maternally inherited 3-kb STX16 deletion, a well-established cause of autosomal dominant PHP-Ib. Although GNAS methylation changes were not detected in additional pediatric and adult patients with subclinical hypothyroidism (23 pediatric and 39 adult cases), hypothyroidism can obviously be the initial finding in PHP-Ib patients. One should therefore consider measuring PTH, along with calcium and phosphate, in patients with unexplained hypothyroidism for extended periods of time to avoid hypocalcemia and associated clinical complications. © 2014 American Society for Bone and Mineral Research © 2014 American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

ABSTRACT Fibrous dysplasia of bone (FD) is a crippling skeletal disease associated with postzygotic mutations (R201C, R201H) of the gene encoding the α subunit of the stimulatory G protein, Gs. By causing a characteristic structural subversion of bone and bone marrow, the disease results in deformity, hypomineralization, and fracture of the affected bones, with severe morbidity arising in childhood or adolescence. Lack of inheritance of the disease in humans is thought to reflect embryonic lethality of germline-transmitted activating Gsα mutations, which would only survive through somatic mosaicism. We have generated multiple lines of mice that express GsαR201C constitutively and develop an inherited, histopathologically exact replica of human FD. Robust transgene expression in neonatal and embryonic tissues and embryonic stem (ES) cells were associated with normal development of skeletal tissues and differentiation of skeletal cells. As in humans, FD lesions in mice developed only in the postnatal life; a defined spatial and temporal pattern characterized the onset and progression of lesions across the skeleton. In individual bones, lesions developed through a sequence of three distinct histopathological stages: a primary modeling phase defined by endosteal/medullary excess bone formation and normal resorption; a secondary phase, with excess, inappropriate remodeling; and a tertiary fibrous dysplastic phase, which reproduced a full-blown replica of the human bone pathology in mice of age ≥1 year. Gsα mutations are sufficient to cause FD, and are per se compatible with germline transmission and normal embryonic development in mice. Our novel murine lines constitute the first model of FD. © 2014 American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

ABSTRACT Pseudohypoparathyroidism type-Ia (PHP-Ia), characterized by renal proximal tubular resistance to parathyroid hormone (PTH), results from maternal mutations of GNAS that lead to loss of α-subunit of the stimulatory G protein (Gαs) activity. Gαs expression is paternally silenced in the renal proximal tubule, and this genomic event is critical for the development of PTH resistance, as patients display impaired hormone action only if the mutation is inherited maternally. The primary clinical finding of PHP-Ia is hypocalcemia, which can lead to various neuromuscular defects including seizures. PHP-Ia patients frequently do not present with hypocalcemia until after infancy, but it has remained uncertain whether PTH resistance occurs in a delayed fashion. Analyzing reported cases of PHP-Ia with documented GNAS mutations and mice heterozygous for disruption of Gnas, we herein determined that the manifestation of PTH resistance caused by the maternal loss of Gαs, ie, hypocalcemia and elevated serum PTH, occurs after early postnatal life. To investigate whether this delay could reflect gradual development of paternal Gαs silencing, we then analyzed renal proximal tubules isolated by laser capture microdissection from mice with either maternal or paternal disruption of Gnas. Our results revealed that, whereas expression of Gαs mRNA in this tissue is predominantly from the maternal Gnas allele at weaning (3 weeks postnatal) and in adulthood, the contributions of the maternal and paternal Gnas alleles to Gαs mRNA expression are equal at postnatal day 3. In contrast, we found that paternal Gαs expression is already markedly repressed in brown adipose tissue at birth. Thus, the mechanisms silencing the paternal Gαs allele in renal proximal tubules are not operational during early postnatal development, and this finding correlates well with the latency of PTH resistance in patients with PHP-Ia. © 2014 American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

Paternally inherited inactivating mutations of the GNAS gene have been associated with a rare and disabling genetic disorder, progressive osseous heteroplasia, in which heterotopic ossification occurs within extraskeletal soft tissues, such as skin, subcutaneous fat, and skeletal muscle. This ectopic bone formation is hypothesized to be caused by dysregulated mesenchymal progenitor cell differentiation that affects a bipotential osteogenic-adipogenic lineage cell fate switch. Interestingly, patients with paternally inherited inactivating mutations of GNAS are uniformly lean. Using a mouse model of Gsα-specific exon 1 disruption, we examined whether heterozygous inactivation of Gnas affects adipogenic differentiation of mesenchymal precursor cells from subcutaneous adipose tissues (fat pad). We found that paternally inherited Gsα inactivation (Gsα+/p−) impairs adipogenic differentiation of adipose-derived stromal cells (ASCs). The Gsα+/p− mutation in ASCs also decreased expression of the adipogenic factors CCAAT-enhancer-binding protein (C/EBP)β, C/EBPα, peroxisome proliferator-activated receptor gamma, and adipocyte protein 2. Impaired adipocyte differentiation was rescued by an adenylyl cyclase activator, forskolin, and provided evidence that Gsα-cAMP signals are necessary in early stages of this process. Supporting a role for Gnas in adipogenesis in vivo, fat tissue weight and expression of adipogenic genes from multiple types of adipose tissues from Gsα+/p− mice were significantly decreased. Interestingly, the inhibition of adipogenesis by paternally inherited Gsα mutation also enhances expression of the osteogenic factors, msh homeobox 2, runt-related transcription factor 2, and osteocalcin. These data support the hypothesis that Gsα plays a critical role in regulating the balance between fat and bone determination in soft tissues, a finding that has important implications for a wide variety of disorders of osteogenesis and adipogenesis. S TEM C ELLS 2012;30:1477-1485 [ABSTRACT FROM AUTHOR]

Fibroblast growth factor-23 (FGF23) is a phosphate- and vitamin D-regulating hormone derived from osteoblasts/osteocytes that circulates in both active (intact, iFGF23) and inactive (C-terminal, cFGF23) forms. O-glycosylation by O-glycosyl transferase N-acetylgalactosaminyltransferase 3 (ppGalNAcT3) and differential cleavage by furin have been shown to be involved in regulating the ratio of active to inactive FGF23. Elevated iFGF23 levels are observed in a number of hypophosphatemic disorders, such as X-linked, autosomal recessive, and autosomal dominant hypophosphatemic rickets, whereas low iFGF23 levels are found in the hyperphosphatemic disorder familial tumoral calcinosis/hyperphosphatemic hyperostosis syndrome. Fibrous dysplasia of bone (FD) is associated with increased total FGF23 levels (cFGF23 + iFGF23); however, classic hypophosphatemic rickets is uncommon. Our results suggest that it can be explained by increased FGF23 cleavage leading to an increase in inactive cFGF23 relative to active iFGF23. Given the fact that FD is caused by activating mutations in the small G-protein Gsα that results in increased cyclic adenosine monophosphate (cAMP) levels, we postulated that there may be altered FGF23 cleavage in FD and that the mechanism may involve alterations in cAMP levels and ppGalNacT3 and furin activities. Analysis of blood specimens from patients with FD confirmed that the elevated total FGF23 levels are the result of proportionally increased cFGF23 levels, consistent with less glycosylation and enhanced cleavage by furin. Analysis of primary cell lines of normal and mutation-harboring bone marrow stromal cells (BMSCs) from patients with FD demonstrated that BMSCs harboring the causative Gsα mutation had higher cAMP levels, lower ppGalNAcT3, and higher furin activity. These data support the model wherein glycosylation by ppGalNAcT3 inhibits FGF23 cleavage by furin and suggest that FGF23 processing is a regulated process that controls overall FGF23 activity in FD patients. © 2012 American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

The article reports on the study conducted to determine the onset, progression and plateau phase of skeletal lesions in fibrous dysplasia, a skeletal disorder that replaces normal bone tissue with abnormal fibro-osseous tissue. Upon studying 109 patients with fibrous dysplasia, it was concluded that most of the lesions occur within the first decade of life and the treatment must be aimed on preventing the progress of the disease.

The article provides information on fibrous dysplasia (FD) of bone as a stem cell disease. Stem cell disease provides a way to generate appropriate models of the disease, which will continue to provide further insight into its natural history and pathogenesis. Moreover, skeletal stem cells can be conceived as directed to change the in vivo behavior of mutated stem cells or to correct the genetic defect in the stem cells themselves.

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. [ABSTRACT FROM AUTHOR]