Gsα Activity Research Articles

SAT-LB67 Pseudohypoparathyroidism 1B Presenting in a Woman Aborting With Multiple Cerebral Calcifications

Background. Pseudohypoparathyroidism is a heterogeneous condition characterized by hypocalcaemia and hyperphosphatemia as a result of the resistance of the target organ to parathyroid hormone (PTH) 1. It is classified into several different entities (1A, 1B, 1C AND 2) according to molecular and clinical causes. PHP1B has resistance to PTH, normal levels of Gsα activity and has no clear signs of hereditary Albright osteodystrophy (AHO) or other hormonal resistance. 2 Clinical case. A 26-year-old woman was admitted to the emergency service due to an abortion and severe hypocalcemia. Background: generalized tonic-clonic seizures and hypocalcemia for nine years without treatment. The physical exam showed Chvostek and Trousseau, and round face. Its height is 148 cm, weight 60 kg, BMI of 27 kg/m2, without clinical features of AHO. Analytics reported Calcium 4.6 mg / dl (8.5-10.5), βHCG 1795 mIU / ml (<10), PTH 206 pg / ml (15-65), phosphorus 7.35 mg / dl (2.5-4.9) Creatinine 0.57 mg / dl, magnesium 2.02 mg / dl, 25-OH-D3 29ng/ml (>30), TSH 4.2uUI /ml (0.27-4.20), Hb 10.6 mg/dl. A brain CT scan showed calcifications in the basal ganglia (thick annular), subcortical area (crescent) and cerebellar hemispheres. Renal and thyroid ultrasound without alterations. Radiographs of four limbs and skull found no radiological signs of AHO. Ophthalmologic evaluation revealed bilateral cataract.During hospitalization she underwent curettage, was treated with calcium gluconate, then calcium and calcitriol supplements. At follow-up, serum and urine calcium levels were monitored for optimal treatment. Conclusions. We report a patient with typical biochemical findings of PHP and in the absence of AHO it would be classified as 1B. The importance of early recognition and a mandatory multidisciplinary approach offer a better prognosis avoiding extensive brain calcifications, seizures and obstetric complications. The long-term treatment of hypocalcemia associated with resistance to PTH is similar but generally more aggressive than that of primary hypoparathyroidism. 1. Linglart A, Levine M, Juppner H. Pseudohypoparathyroidism. Endocrinol Metab Clin North Am 2018; 47: 865-888. 2. Mantovani G, Bastepe M, et al. Diagnosis and treatment of pseudohypoparathyroidism and related disorders: first declaration of international consensus. Nat Rev Endocrinol. 2018; 14 (8): 476-500.

Pseudohypoparathyroidism.

The term pseudohypoparathyroidism (PHP) refers to a spectrum of rare disorders of mineral metabolism, characterized by features due to end-organ resistance to PTH. The phenotypes of Albright hereditary osteodystrophy (AHO), originally described as associated to the disease, and progressive osseous heteroplasia, can be associated to the endocrine manifestations of hormonal resistance. Genetic or epigenetic alterations in the complex imprinted GNAS locus, encoding the alpha-subunit of the stimulatory G protein (GSα) and several other transcripts, give rise to the different forms oh PHP, which can be differentiated according to the phenotype, the response to PTH infusion and in vitro assays testing Gsα activity. Since PHP-related phenotypes are overlapping and other non GNAS-dependent disorders mimicking AHO, such as acrodysostosis, have been genetically characterized, the term PHP is today considered obsolete and better referred to the more comprehensive "inactivating PTH/PTHrP signaling disorder (iPPSD)" as proposed in a recent classification. This broad term include all the congenital rare disorders due to impaired PTH/PTHrP cAMP pathway. Genetic and epigenetic analyses, although not necessary for diagnosis made on the basis of major and minor criteria according to clinical and biochemical signs, will let to differentiate among the different forms for proper therapeutic planning, counseling and follow-up.

Constitutive stimulatory G protein activity in limb mesenchyme impairs bone growth.

GNAS mutations leading to constitutively active stimulatory G protein alpha-subunit (Gsα) cause different tumors, fibrous dysplasia of bone, and McCune-Albright syndrome, which are typically not associated with short stature. Enhanced signaling of the parathyroid hormone/parathyroid hormone-related peptide receptor, which couples to multiple G proteins including Gsα, leads to short bones with delayed endochondral ossification. It has remained unknown whether constitutive Gsα activity also impairs bone growth. Here we generated mice expressing a constitutively active Gsα mutant (Gsα-R201H) conditionally upon Cre recombinase (cGsαR201H mice). Gsα-R201H was expressed in cultured bone marrow stromal cells from cGsαR201H mice upon adenoviral-Cre transduction. When crossed with mice in which Cre is expressed in a tamoxifen-regulatable fashion (CAGGCre-ER™), tamoxifen injection resulted in mosaic expression of the transgene in double mutant offspring. We then crossed the cGsαR201H mice with Prx1-Cre mice, in which Cre is expressed in early limb-bud mesenchyme. The double mutant offspring displayed short limbs at birth, with narrow hypertrophic chondrocyte zones in growth plates and delayed formation of secondary ossification center. Consistent with enhanced Gsα signaling, bone marrow stromal cells from these mice demonstrated increased levels of c-fos mRNA. Our findings indicate that constitutive Gsα activity during limb development disrupts endochondral ossification and bone growth. Given that Gsα haploinsufficiency also leads to short bones, as in patients with Albright's hereditary osteodystrophy, these results suggest that a tight control of Gsα activity is essential for normal growth plate physiology.

Follow-up Findings in a Turkish Girl with Pseudohypoparathyroidism Type Ia Caused by a Novel Heterozygous Mutation in the GNAS Gene

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, Gsα activity. To our knowledge, this is the first case report describing bioinactive growth hormone in PHP-Ia.

A Novel T55A Variant of Gs α Associated with Impaired cAMP Production, Bone Fragility, and Osteolysis.

G-protein coupled receptors (GPCRs) mediate a wide spectrum of biological activities. The GNAS complex locus encodes the stimulatory alpha subunit of the guanine nucleotide binding protein (Gsα) and regulates production of the second messenger cyclic AMP (cAMP). Loss-of-function GNAS mutations classically lead to Albright's Hereditary Osteodystrophy (AHO) and pseudohypoparathyroidism, often with significant effects on bone formation and mineral metabolism. We present the case of a child who exhibits clinical features of osteolysis, multiple childhood fractures, and neonatal SIADH. Exome sequencing revealed a novel de novo heterozygous missense mutation of GNAS (c.163A<G, p.T55A) affecting the p-loop of the catalytic Gsα GTPase domain. In order to further assess whether this unique mutation resulted in a gain or loss of function of Gsα, we introduced the mutation into a rat GNAS plasmid and performed functional studies to assess the level of cAMP activity associated with this mutation. We identified a 64% decrease in isoproterenol-induced cAMP production in vitro, compared to wild type, consistent with loss of Gsα activity. Despite a significant decrease in isoproterenol-induced cAMP production in vitro, this mutation did not produce a classical AHO phenotype in our patient; however, it may account for her presentation with childhood fractures and osteolysis.

Evidence of hormone resistance in a pseudo-pseudohypoparathyroidism patient with a novel paternal mutation in GNAS

ContextLoss-of-function GNAS mutations lead to hormone resistance and Albright's hereditary osteodystrophy (AHO) when maternally inherited, i.e. pseudohypoparathyroidism-Ia (PHPIa), but cause AHO alone when located on the paternal allele, i.e. pseudoPHP (PPHP). ObjectiveWe aimed to establish the molecular diagnosis in a patient with AHO and evidence of hormone resistance. CaseThe patient is a female who presented at the age of 13.5years with short stature and multiple AHO features. No evidence for TSH or gonadotropin-resistance was present. Serum calcium and vitamin D levels were normal. However, serum PTH was elevated on multiple occasions (64–178pg/mL, normal: 9–52) and growth hormone response to clonidine or L-DOPA was blunted, suggesting hormone resistance and PHP-Ia. The patient had diminished erythrocyte Gsα activity and a novel heterozygous GNAS mutation (c.328 G>C; p.A109P). The mother lacked the mutation, and the father's DNA was not available. Hence, a diagnosis of PPHP also appeared possible, supported by low birth weight and a lack of AHO features associated predominantly with PHP-Ia, i.e. obesity and cognitive impairment. To determine the parental origin of the mutation, we amplified the paternally expressed A/B and biallelically expressed Gsα transcripts from the patient's peripheral blood RNA. While both wild-type and mutant nucleotides were detected in the Gsα amplicon, only the mutant nucleotide was present in the A/B amplicon, indicating that the mutation was paternal. ConclusionThese findings suggest that PTH and other hormone resistance may not be an exclusive feature of PHP-Ia and could also be observed in patients with PPHP.

Guanine Nucleotide-Binding Protein α Subunit Hypofunction in Children with Short Stature and Disproportionate Shortening of the 4th and 5th Metacarpals

Background:GNAS encodes the α subunit of the stimulatory G protein (Gsα). Maternal inherited Gsα mutations cause pseudohypoparathyroidism type Ia (PHP-Ia), associated with shortening of the 4th and 5th metacarpals. Aims: Here we investigated the Gsα pathway in short patients with distinct shortening of the 4th and 5th metacarpals. Methods: In 571 children with short stature and 4 patients with PHP-Ia metacarpal bone lengths were measured. In identified patients we analysed the Gsα protein function in platelets, performed GNAS sequencing, and epigenetic analysis of four significant differentially methylated regions. Results: In 51 patients (8.9%) shortening of the 4th and 5th metacarpals was more pronounced than their height deficit. No GNAS coding mutations were identified in 20 analysed patients, except in 2 PHP-Ia patients. Gsα activity was reduced in all PHP-Ia patients and in 25% of the analysed patients. No significant methylation changes were identified. Conclusions: Our findings suggest that patients with short stature and distinct metacarpal bone shortening could be part of the wide variety of PHP/PPHP, therefore it was worthwhile analysing the Gsα protein function and GNAS gene in these patients in order to further elucidate the phenotype and genotype of Gsα dysfunction.

Aspectos clínicos en dos casos de seudohipoparatiroidismo (ia y ib) y estudio molecular del locus GNAS

Pseudohypoparathyroidism (PHP) is a rare disorder, characterized by a tissue resistance to parathyroid hormone (PTH). The two main subtypes of PHP, PHPIa and PHPIb, are caused by alterations in the GNAS locus (20q13.3), which encodes the Gsα protein, essential for the action of PTH and other hormones.PHP-Ia is associated with several hormone resistances, Albright hereditary osteodystrophy (AHO), and reduced Gsα activity. It is caused by inactivating mutations in the GNAS gene. PHP-Ib presents with isolated resistance to PTH, without AHO and with normal to low Gsα activity. It is related to imprinting defects in GNAS.Two unrelated cases of PHP-Ia and PHP-Ib are presented here, focusing on their clinical aspects and in the differential diagnosis with similar pathologies.

GNAS Epigenetic Defects and Pseudohypoparathyroidism: Time for a New Classification?

The two main subtypes of pseudohypoparathyroidism (PHP), PHP-Ia and -Ib, are caused by mutations in GNAS exons 1–13 and methylation defects in the imprinted GNAS cluster, respectively. PHP-Ia patients show Albright hereditary osteodystrophy (AHO), together with end-organ resistance to the action of different hormones that activate the Gs-coupled pathways, primarily PTH, TSH, gonadotropins, and GHRH. In PHP-Ib patients AHO is classically absent, Gsα activity in erythrocytes and fibroblast is normal and hormone resistance is limited to PTH and TSH, the latter being essentially always subclinical. This disorder is caused by the disruption of long-range imprinting control elements in GNAS locus. In particular, the most consistent defect is the loss of imprinting at the exon A/B differenzially methylated region (DMR), with consequent decreased Gsα transcription in tissues where this protein is derived from the maternal allele only. The familial form of the disease (AD-PHP-Ib) is typically associated with an isolated loss of imprinting at the exon A/B DMR due to microdeletions disrupting the upstream STX16 gene. In addition, deletions removing the entire NESP55 DMR, located within GNAS, have been identified in some AD-PHP-Ib kindreds in whom affected individuals show loss of all the maternal GNAS imprints. Conversely, most sporadic PHP-Ib cases have GNAS imprinting abnormalities that involve multiple DMRs, but the genetic lesion underlying these defects remains to be discovered. Recently, methylation defects have been detected in a subset of patients with PHP-Ia and variable degrees of AHO, indicating a molecular overlap between the two forms. Imprinting defects do not seem to be associated with the severity of AHO neither with specific AHO signs.

Gsα activity is reduced in erythrocyte membranes of patients with psedohypoparathyroidism due to epigenetic alterations at the GNAS locus

In pseudohypoparathyroidism (PHP), PTH resistance results from impairment of signal transduction of G protein-coupled receptors caused by a deficiency of the Gsα-cAMP signaling cascade due to diminished Gsα activity in maternally imprinted tissues. In PHP-Ia, inactivating mutations of the GNAS gene lead to haploinsufficiency in some tissues with biallelic expression, so in addition to PHP, Albright's hereditary osteodystrophy (AHO) is also present. In PHP-Ib, caused by methylation defects at the GNAS locus, diminished Gsα activity was thought to be limited to maternally imprinted tissues, such as the renal proximal tubule and the thyroid, leading to a lack of AHO. Recently, we demonstrated methylation defects in patients with AHO signs, indicating a connection between epigenetic changes and AHO. Our objective was to determine Gsα activity in erythrocyte membranes in patients with epigenetic defects at the GNAS locus compared to normal controls and patients with inactivating GNAS mutations. Gsα activity and expression, mutation of the GNAS locus, and methylation status were studied in patients with PHP and mild signs of AHO (PHP-Ia: 12; PHP-Ib: 17, of which 8 had some features of AHO). Then, we statistically compared the Gsα activity of the different PHP subtypes. Patients with methylation defects at the GNAS locus show a significant decrease in erythrocyte Gsα activity compared to normal controls (PHP-Ib versus controls, p < .001). This was significantly lower in patients with AHO signs (PHP-Ib + mild-AHO versus PHP-Ib, p < .05). Our research shows that PHP-Ia and PHP-Ib classification is not only overlapped genetically, as reported, but also in terms of Gsα activity. Reduced expression of GNAS due to methylation defects could downregulate Gsα activity in other tissues beyond those described and could also be causative of AHO.

A Disruptive Mutation in Exon 3 of the GNAS Gene with Albright Hereditary Osteodystrophy, Normocalcemic Pseudohypoparathyroidism, and Selective Long Transcript Variant Gsα-L Deficiency

The GNAS gene encodes the alpha-subunit of stimulatory G proteins, which play a crucial role in intracellular signal transduction of peptide and neurotransmitter receptors. In addition to transcript variants that differ in their first exon due to different promoters, there are two long (Gsalpha-L) and two short (Gsalpha-S) splice variants, created by alternative splicing. Heterozygous inactivating maternally inherited mutations of GNAS lead to a phenotype in which Albright hereditary osteodystrophy is associated with pseudohypoparathyroidism type Ia. The GNAS gene of a 10-yr-old girl with brachymetacarpia, mental retardation, normocalcemic pseudohypoparathyroidism, and hypothyroidism was investigated. We found a heterozygous insertion of an adenosine in exon 3 altering codon 85 and leading to a frame shift inducing a stop codon in exon 4. Molecular studies of cDNA from blood RNA demonstrated normal, biallelic expression of Gsalpha-S transcripts, whereas expression of Gsalpha-L transcripts from the maternal allele was reduced. Immunoblot analysis revealed a reduced Gsalpha-L protein level to about 50%, whereas the protein level of Gsalpha-S was unaltered. Furthermore, the Gsalpha protein activity in erythrocyte membranes was diminished to about 75% of normal. Both the reduced activity and the mutation were also found in the mother and the affected younger brother. This report demonstrates the first evidence for a pathogenic mutation in exon 3 of the GNAS gene. The mutation is associated with a phenotype of Albright hereditary osteodystrophy and pseudohypoparathyroidism type Ia due to selective deficiency of Gsalpha-L and a partial reduction of Gsalpha activity.

Alterations of adenylyl cyclase and G proteins in aortocaval shunt-induced heart failure.

Unlike most other experimental models of congestive heart failure, the volume overload model induced by aortocaval shunt (AVS) in rats was found to exhibit enhanced beta-adrenoceptor (beta-AR) signaling. To study whether the adenylyl cyclase (AC)-G protein system is involved in such a change, we examined cardiac AC activity and protein content as well as G(s)alpha and G(i)alpha activities, protein contents, and mRNA levels in both left (LV) and right (RV) ventricles at the failing stage (16 wk after surgery). Basal and forskolin-stimulated AC activities were significantly increased in both LV and RV from the failing hearts; this change was associated with an upregulation of type V/VI AC protein. In contrast to 5'-guanylyl imidodiphosphate and NaF, the stimulatory effect of isoproterenol on AC was increased in the failing heart. Although G(s)alpha and G(i)alpha protein contents in the failing hearts were not altered, the mRNA level for G(s)alpha was decreased by 20% and that for G(i)alpha was increased by 20%. In addition, the activity of G(s)alpha, but not G(i)alpha, as assessed by toxin-catalyzed ADP ribosylation, was significantly decreased in the failing heart. Losartan and imidapril treatments improved cardiac function and attenuated alterations in mRNA levels for G(s)alpha and G(i)alpha proteins, as well as G(s)alpha activity, without affecting changes in AC protein content or activities in heart failure due to volume overload. These data suggest that increased AC activity may contribute to the enhanced beta-AR signaling in the AVS model of heart failure, whereas alterations in gene expression for G proteins may be of an adaptive nature at this stage of heart failure.

Pseudohypoparathyroidism: new insight into Gsα

Pseudohypoparathyroidism (PHP) is an endocrinopathy characterized by resistance to parathyroid hormone (PTH) in target tissues, resulting in hypocalcaemia and hyperphosphataemia despite increased secretion of PTH. PHP is subclassified according to additional biochemical and clinical criteria. In PHP type Ia, patients commonly show resistance to thyroid stimulating hormone (TSH) and luteinizing hormone (LH), as well as to PTH, and have clinical features known as Albright hereditary osteodystrophy (AHO) characterized by round faces, short stature and shortened fingers and toes. At the molecular level, PHPIa is known to result from heterozygous inactivating mutations in the gene GNAS1, which encodes the α-subunit of the heterotrimeric G-protein, Gs. Gs couples the receptors for various hormones, including PTH, TSH and LH, to activation of intracellular adenylyl cyclase. Patients with PHPIa show a 50% reduction in Gsα activity in their cell membranes.By contrast, PHP type Ib is characterized by isolated resistance to PTH, normal Gsα activity and absence of AHO. It was originally hypothesized that PHPIb would result from mutations of the PTH receptor gene, but extensive analysis failed to confirm this. Recently, PHPIb was localized to a region of chromosome 20 very close to GNAS1. Wu et al. 1xSee all References1 now describe an intriguing novel mutation in GNAS1 that leads to PHPIb rather than PHPIa. The mutation was identified in three affected brothers and results in deletion of one amino acid at the C-terminus of Gsα, a region important for receptor interaction. In vitro expression assays confirmed that the mutation selectively uncouples Gsα from the PTH receptor without affecting its interaction with receptors for TSH or LH, thus explaining the PTH-specific phenotype. The mutation was not found in 15 additional unrelated patients and is therefore not a common cause of PHPIb. Other possible mechanisms include promoter mutations with tissue-specific effects, or mutations in alternative Gsα transcripts. GNAS1 is already known to be a complex imprinted locus and this report underlines the molecular complexities of Gsα expression and function.

Pseudohypoparathyroidism: from bedside to bench and back.

Pseudohypoparathyroidism: from bedside to bench and back.