Functional characterization of GNAS mutations found in patients with pseudohypoparathyroidism type Ic defines a new subgroup of pseudohypoparathyroidism affecting selectively Gs?-receptor interaction.
ABSTRACT: Pseudohypoparathyroidism type Ia (PHPIa) is caused by GNAS mutations leading to deficiency of the ?-subunit of stimulatory G proteins (Gs?) that mediate signal transduction of G protein-coupled receptors via cAMP. PHP type Ic (PHPIc) and PHPIa share clinical features of Albright hereditary osteodystrophy (AHO); however, in vitro activity of solubilized Gs? protein is normal in PHPIc but reduced in PHPIa. We screened 32 patients classified as PHPIc for GNAS mutations and identified three mutations (p.E392K, p.E392X, p.L388R) in four unrelated families. These and one novel mutation associated with PHPIa (p.L388P) were introduced into a pcDNA3.1(-) expression vector encoding Gs? wild-type and expressed in a Gs?-null cell line (Gnas(E2-/E2-) ). To investigate receptor-mediated cAMP accumulation, we stimulated the endogenous expressed ?(2) -adrenergic receptor, or the coexpressed PTH or TSH receptors, and measured the synthesized cAMP by RIA. The results were compared to receptor-independent cholera toxin-induced cAMP accumulation. Each of the mutants associated with PHPIc significantly reduced or completely disrupted receptor-mediated activation, but displayed normal receptor-independent activation. In contrast, PHPIa associated p.L388P disrupted both receptor-mediated activation and receptor-independent activation. We present a new subgroup of PHP that is caused by Gs? deficiency and selectively affects receptor coupling functions of Gs?.
Project description:Patients diagnosed with pseudohypoparathyroidism type Ia (PHP Ia) suffer from hormonal resistance and abnormal postural features, in a condition classified as Albright hereditary osteodystrophy (AHO) syndrome. This syndrome is linked to a maternally inherited mutation in the GNAS complex locus, encoding for the GTPase subunit Gs?. Here, we investigated how platelet phenotype and omics analysis can assist in the often difficult diagnosis. By coupling to the IP receptor, Gs? induces platelet inhibition via adenylyl cyclase and cAMP-dependent protein kinase A (PKA). In platelets from seven patients with suspected AHO, one of the largest cohorts examined, we studied the PKA-induced phenotypic changes. Five patients with a confirmed GNAS mutation, displayed impairments in Gs?-dependent VASP phosphorylation, aggregation, and microfluidic thrombus formation. Analysis of the platelet phosphoproteome revealed 2,516 phosphorylation sites, of which 453 were regulated by Gs?-PKA. Common changes in the patients were: (1) a joint panel of upregulated and downregulated phosphopeptides; (2) overall PKA dependency of the upregulated phosphopeptides; (3) links to key platelet function pathways. In one patient with GNAS mutation, diagnosed as non-AHO, the changes in platelet phosphoproteome were reversed. This combined approach thus revealed multiple phenotypic and molecular biomarkers to assist in the diagnosis of suspected PHP Ia.
Project description:Disorders related to parathyroid hormone (PTH) resistance and PTH signaling pathway impairment are historically classified under the term of pseudohypoparathyroidism (PHP). The disease was first described and named by Fuller Albright and colleagues in 1942. Albright hereditary osteodystrophy (AHO) is described as an associated clinical entity with PHP, characterized by brachydactyly, subcutaneous ossifications, round face, short stature and a stocky build. The classification of PHP is further divided into PHP-Ia, pseudo-PHP (pPHP), PHP-Ib, PHP-Ic and PHP-II according to the presence or absence of AHO, together with an in vivo response to exogenous PTH and the measurement of Gs? protein activity in peripheral erythrocyte membranes in vitro. However, PHP classification fails to differentiate all patients with different clinical and molecular findings for PHP subtypes and classification become more complicated with more recent molecular characterization and new forms having been identified. So far, new classifications have been established by the EuroPHP network to cover all disorders of the PTH receptor and its signaling pathway. Inactivating PTH/PTH-related protein signaling disorder (iPPSD) is the new name proposed for a group of these disorders and which can be further divided into subtypes - iPPSD1 to iPPSD6. These are termed, starting from PTH receptor inactivation mutation (Eiken and Blomstrand dysplasia) as iPPSD1, inactivating Gs? mutations (PHP-Ia, PHP-Ic and pPHP) as iPPSD2, loss of methylation of GNAS DMRs (PHP-Ib) as iPPSD3, PRKAR1A mutations (acrodysostosis type 1) as iPPSD4, PDE4D mutations (acrodysostosis type 2) as iPPSD5 and PDE3A mutations (autosomal dominant hypertension with brachydactyly) as iPPSD6. iPPSDx is reserved for unknown molecular defects and iPPSDn+1 for new molecular defects which are yet to be described. With these new classifications, the aim is to clarify the borders of each different subtype of disease and make the classification according to molecular pathology. The iPPSD group is designed to be expandable and new classifications will readily fit into it as necessary.
Project description:Pseudohypoparathyroidism type Ia (PHP-Ia) is characterized by multihormone resistance and an Albright hereditary osteodystrophy (AHO) phenotype. It is caused by heterozygous mutations in GNAS gene. Clinical and biochemical findings of a female PHP-Ia patient were evaluated from age of diagnosis (6.5 years) to 14.5 years of age. The patient had short stature, brachydactyly, and subcutaneous heterotopic ossifications. Serum calcium and phosphorus levels were normal, but parathyroid hormone levels were high. Based on the typical clinical findings of AHO phenotype and biochemical findings, she was diagnosed as having PHP-Ia. A novel heterozygous mutation (c.128T>C) was found in the GNAS gene. Follow-up examinations revealed resistance to thyroid-stimulating hormone and a bioinactive growth hormone. Clinicians should take into consideration PHP-Ia in patients referred with short stature, and patients with an AHO phenotype must be further evaluated for hormone resistance, GNAS gene mutation, Gs? activity. To our knowledge, this is the first case report describing bioinactive growth hormone in PHP-Ia.
Project description:Pseudohypoparathyroidism type I (PHP-I) is divided into PHP-Ia with Albright hereditary osteodystrophy and PHP-Ib, which usually shows no Albright hereditary osteodystrophy features. Although PHP-Ia and PHP-Ib are typically caused by genetic defects involving ? subunit of the stimulatory G protein (Gs?)-coding GNAS exons and methylation defects of the GNAS differentially methylated regions (DMRs) on the maternal allele, respectively, detailed phenotypic characteristics still remains to be examined.To clarify phenotypic characteristics according to underlying (epi)genetic causes.We performed (epi)genotype-phenotype analysis in 69 Japanese patients with PHP-I; that is, 28 patients with genetic defects involving Gs?-coding GNAS exons (group 1) consisting of 12 patients with missense variants (subgroup A) and 16 patients with null variants (subgroup B), as well as 41 patients with methylation defects (group 2) consisting of 21 patients with broad methylation defects of the GNAS-DMRs (subgroup C) and 20 patients with an isolated A/B-DMR methylation defect accompanied by the common STX16 microdeletion (subgroup D).Although (epi)genotype-phenotype findings were grossly similar to those reported previously, several important findings were identified, including younger age at hypocalcemic symptoms and higher frequencies of hyperphosphatemia in subgroup C than in subgroup D, development of brachydactyly in four patients of subgroup C, predominant manifestation of subcutaneous ossification in subgroup B, higher frequency of thyrotropin resistance in group 1 than in group 2, and relatively low thyrotropin values in four patients with low T4 values and relatively low luteinizing hormone/follicle-stimulating hormone values in five adult females with ovarian dysfunction.The results imply the presence of clinical findings characteristic of each underlying cause and provide useful information on the imprinting status of Gs?.
Project description:To provide the reader with a review of contemporary literature describing the evolving understanding of the molecular pathobiology of pseudohypoparathyroidism (PHP).The features of PHP type 1 reflect imprinting of the GNAS gene, which encodes the ? subunit of the heterotrimeric G protein (G?(s)) that couples heptahelical receptors to activation of adenylyl cyclase. Transcription of G?(s) is biallelic in most cells, but is primarily from the maternal allele in some tissues (e.g. proximal renal tubules, thyroid, pituitary somatotropes, gonads). Patients with PHP 1a have heterozygous mutations within the exons of the maternal GNAS allele that encode G?(s), whereas patients with PHP 1b have methylation defects in the GNAS locus that reduce transcription of G?(s) from the maternal allele. In both PHP 1a and PHP 1b, paternal imprinting of G?(s) leads to resistance to parathyroid hormone and TSH. Although brachydactyly is characteristic of PHP 1a, it is sometimes present in patients with PHP 1b.Molecular studies enable a distinction between PHP 1a and PHP 1b, with different mechanisms accounting for G?(s) deficiency. Clinical overlap between these two forms of PHP type 1 is likely due to the variable levels of G?(s) activity expressed in specific cell types.
Project description:Stimulatory heterotrimeric G protein (Gs) transduces signals from various cell-surface receptors to adenylyl cyclases, which generate cAMP. The alpha subunit of Gs (Gsalpha) is encoded by GNAS (Gnas in mice), and heterozygous Gsalpha inactivating mutations lead to Albright hereditary osteodystrophy. The in vivo role of Gsalpha in skeletogenesis is largely unknown, because of early embryonic lethality of mice with disruption of Gnas exon 2 (Gnas(E2-/E2-)) and the absence of easily detectable phenotypes in growth plate chondrocytes of heterozygous mutant mice (Gnas(+/E2-)). We generated chimeric mice containing wild-type cells and either Gnas(E2-/E2-) or Gnas(+/E2-) cells. Gnas(E2-/E2-) chondrocytes phenocopied PTH/PTHrP receptor (PPR)(-/-) cells by prematurely undergoing hypertrophy. Introduction of a transgene expressing Gsalpha, one of several gene products that include Gnas exon 2, into Gnas(E2-/E2-) cells prevented premature hypertrophy. Gsalpha mRNA expression detected by real-time RT-PCR analysis was reduced to approximately half that of the wild-type in both paternal and maternal Gnas(+/E2-) growth plate chondrocytes, indicating biallelic expression of Gsalpha in these cells. Hypertrophy of Gnas(+/E2-) chondrocytes was modestly but significantly premature in chimeric growth plates of mice containing wild-type and Gnas(+/E2-) cells. These data suggest that Gsalpha is the primary mediator of the actions of PPR in growth plate chondrocytes and that there is haploinsufficiency of Gsalpha signaling in Gnas(+/E2-) chondrocytes.
Project description:BACKGROUND:Pseudohypoparathyroidism type 1A (PHP1A) is a rare genetic disease primarily characterized by resistance to parathyroid hormone along with hormonal resistance and other features of Albright hereditary osteodystrophy (AHO). It is caused by heterozygous inactivating mutations in the maternal allele of the GNAS gene, which encodes the stimulatory G-protein alpha subunit (Gs?) and regulates production of the second messenger cyclic AMP (cAMP). Herein, we report a case of of PHP1A with atypical clinical manifestations (oligomenorrhea, subclinical hypothyroidism, and normocalcemia) and explore the underlying genetic cause in this patient. METHODS:Blood samples were collected from the patient, her family members, and 100 healthy controls. The 13 exons and flanking splice sites of the GNAS gene were amplified by PCR and sequenced. To further assess whether the novel mutation resulted in gain or loss of function of Gs?, we examined the level of cAMP activity associated with this mutation through in vitro functional studies by introducing the target mutation into a human GNAS plasmid. RESULTS:A novel heterozygous c.715A?>?G (p.N239D) mutation in exon 9 of the GNAS gene was identified in the patient. This mutation was also found in her mother, who was diagnosed with pseudopseudohypoparathyroidism. An in vitro cAMP assay showed a significant decrease in PTH-induced cAMP production in cells transfected with the mutant plasmid, compared to that in the wild-type control cells (P?<?0.01), which was consistent with loss of Gsa activity. CONCLUSION:We identified a novel GNAS mutation that altered Gs? function, which furthers our understanding of the pathogenesis of this disease. Screening for GNAS mutations should be considered in suspected cases of PHP1A even if the classical signs are not present.
Project description:Several endocrine diseases that share resistance to PTH are grouped under the term pseudohypoparathyroidism (PHP). Patients with PHP type Ia show additional hormone resistance, defective erythrocyte G(s)alpha activity, and dysmorphic features termed Albright's hereditary osteodystrophy (AHO). Patients with PHP-Ib show less diverse hormone resistance and normal G(s)alpha activity; AHO features are typically absent in PHP-Ib. Mutations affecting G(s)alpha coding exons of GNAS and epigenetic alterations in the same gene are associated with PHP-Ia and -Ib, respectively. The epigenetic GNAS changes in familial PHP-Ib are caused by microdeletions near or within GNAS but without involving G(s)alpha coding exons.We sought to identify the molecular defect in a patient who was diagnosed with PHP-Ia based on clinical presentation (hormone resistance and AHO) but displayed the molecular features typically associated with PHP-Ib (loss of methylation at exon A/B) without previously described genetic mutations.Microsatellite typing, comparative genome hybridization, and allelic dosage were performed for proband and her parents.Comparative genome hybridization revealed a deletion of 30,431 bp extending from the intronic region between exons XL and A/B to intron 5. The same mutation was also demonstrated, by PCR, in the patient's mother, but polymorphism and allele dosage analyses indicated that she had this mutation in a mosaic manner.We discovered a novel multiexonic GNAS deletion transmitted to our patient from her mother who is mosaic for this mutation. The deletion led to different phenotypic manifestations in the two generation and appeared, in the patient, as loss of GNAS imprinting.
Project description:Pseudohypoparathyroidism (PHP) types 1a and 1b are distinguished by clinical, biochemical, and molecular features. We report extended kindred with PHP 1b in which many affected members also had growth plate defects, including brachydactyly and a Madelung-like deformity.Analyses included clinical examination, assessment of mineral metabolism, thyroid function, skeletal radiography, and analysis of the GNAS and STX16 genes.Patients were studied in an academic medical center.We studied 37 members of a family in which PHP 1b occurred in 23 individuals. Ten of 17 affected patients who were examined had brachydactyly E, including two subjects with Madelung-like defects. Five of 16 subjects had subclinical hypothyroidism; no subject showed sc ossification or short stature. None of the unaffected members had brachydactyly or an elevated serum level of PTH or TSH. Levels of immunoactive erythrocyte G?(s) were normal in two affected subjects tested. Linkage analysis indicated linkage between PTH resistance and the GNAS gene locus; however, no mutations were identified in GNAS exons 1-13. Methylation analysis of genomic DNA from affected subjects showed loss of maternal epigenotype in exon 1A with normal methylation of the differentially methylated regions for XLG?s and NESP55, and PCR demonstrated heterozygosity for a 3.0-kb deletion in the STX16 gene.The segregation of brachydactyly with PHP 1b in this family indicates that an imprinting defect in GNAS can lead to growth plate defects, including brachydactyly and Madelung deformity. These features suggest that GNAS signaling plays a more extensive role in chondrocyte maturation than previously thought.
Project description:Pseudohypoparathyroidism (PHP) is caused by (epi)genetic defects in the imprinted GNAS cluster. Current classification of PHP patients is hampered by clinical and molecular diagnostic overlaps. The European Consortium for the study of PHP designed a genome-wide methylation study to improve molecular diagnosis.The HumanMethylation 450K BeadChip was used to analyze genome-wide methylation in 24 PHP patients with parathyroid hormone resistance and 20 age- and gender-matched controls. Patients were previously diagnosed with GNAS-specific differentially methylated regions (DMRs) and include 6 patients with known STX16 deletion (PHP(?stx16)) and 18 without deletion (PHP(neg)).The array demonstrated that PHP patients do not show DNA methylation differences at the whole-genome level. Unsupervised clustering of GNAS-specific DMRs divides PHP(?stx16) versus PHP(neg) patients. Interestingly, in contrast to the notion that all PHP patients share methylation defects in the A/B DMR while only PHP(?stx16) patients have normal NESP, GNAS-AS1 and XL methylation, we found a novel DMR (named GNAS-AS2) in the GNAS-AS1 region that is significantly different in both PHP(?stx16) and PHP(neg), as validated by Sequenom EpiTYPER in a larger PHP cohort. The analysis of 58 DMRs revealed that 8/18 PHP(neg) and 1/6 PHP(?stx16) patients have multi-locus methylation defects. Validation was performed for FANCC and SVOPL DMRs.This is the first genome-wide methylation study for PHP patients that confirmed that GNAS is the most significant DMR, and the presence of STX16 deletion divides PHP patients in two groups. Moreover, a novel GNAS-AS2 DMR affects all PHP patients, and PHP patients seem sensitive to multi-locus methylation defects.