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

Pseudohypoparathyroidism-Ia and -Ib (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 resistance to the action of different hormones that activate the Gs-coupled pathway. In PHP-Ib patients AHO is classically absent and hormone resistance is limited to PTH and TSH. This disorder is caused by GNAS methylation alterations with loss of imprinting at the exon A/B differentially methylated region (DMR) being the most consistent and recurrent defect. 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, associated with loss of all the maternal GNAS imprints have been identified in some AD-PHP-Ib kindreds. Conversely, most sporadic PHP-Ib cases have GNAS imprinting abnormalities that involve multiple DMRs, but the genetic lesion underlying these defects is unknown. 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 2 forms. Imprinting defects do not seem to be associated with the severity of AHO neither with specific AHO signs. In conclusion, the latest findings on the molecular basis underlying these defects suggest the existence of a clinical and genetic/epigenetic overlap between PHP-Ia and PHP-Ib, and highlight the necessity of a new clinical classification of these disorders based on molecular findings.     

Paternal uniparental isodisomy of chromosome 20q--and the resulting changes in GNAS1 methylation--as a plausible cause of pseudohypoparathyroidism

Heterozygous inactivating mutations in the GNAS1 exons (20q13.3) that encode the alpha-subunit of the stimulatory G protein (Gsalpha) are found in patients with pseudohypoparathyroidism type Ia (PHP-Ia) and in patients with pseudo-pseudohypoparathyroidism (pPHP). However, because of paternal imprinting, resistance to parathyroid hormone (PTH)-and, sometimes, to other hormones that require Gsalpha signaling-develops only if the defect is inherited from a female carrier of the disease gene. An identical mode of inheritance is observed in kindreds with pseudohypoparathyroidism type Ib (PHP-Ib), which is most likely caused by mutations in regulatory regions of the maternal GNAS1 gene that are predicted to interfere with the parent-specific methylation of this gene. We report a patient with PTH-resistant hypocalcemia and hyperphosphatemia but without evidence for Albright hereditary osteodystrophy who has paternal uniparental isodisomy of chromosome 20q and lacks the maternal-specific methylation pattern within GNAS1. Since studies in the patient's fibroblasts did not reveal any evidence of impaired Gsalpha protein or activity, it appears that the loss of the maternal GNAS1 gene and the resulting epigenetic changes alone can lead to PTH resistance in the proximal renal tubules and thus lead to impaired regulation of mineral-ion homeostasis.     

Genetic basis for resistance to parathyroid hormone

Pseudohypoparathyroidism (PHP) is associated with biochemical hypoparathyroidism (i.e. hypocalcemia and hyperphosphatemia) due to parathyroid hormone (PTH) resistance rather than to PTH deficiency. Patients with PHP type 1a have a generalized form of hormone resistance plus a constellation of developmental defects termed Albright hereditary osteodystrophy (AHO). Within PHP type 1a families some individuals will show AHO but have normal hormone responsiveness, a variant phenotype termed pseudo-PHP. By contrast, patients with PHP type 1b manifest only PTH resistance and lack features of AHO. These various forms of PHP are due to defects in the GNAS1 gene that lead to decreased expression or activity of the alpha-subunit of the stimulatory G protein (G(s)alpha). Tissue-specific genomic imprinting of GNAS1 accounts for the variable phenotypes of patients with GNAS1 defects.     

Clinical and biological heterogeneity in pseudohypoparathyroidism syndrome. Results of a multicenter study

Pseudohypoparathyroidism (PHP) is a rare inherited syndrome frequently associated with Albright's hereditary osteodystrophy (AHO). We conducted a multicenter study including 71 PHP children and 77 relatives. Erythrocyte Gsalpha biological activity was measured in each patient (normal range 85-110%). 61 patients were classified into four subtypes based on clinical and endocrine data and Gsalpha activity: 45 PHP Ia, 8 PHP Ib, 2 PHP II, and 6 PHP Ic. PHP Ia had decreased Gsalpha (58 +/- 9%), PHP Ib patients had PTH resistance, no AHO and normal Gsalpha (96 +/- 9%), PHP Ic patients had PTH resistance, AHO and no decreased Gsalpha (97 +/- 13%). The 10 remaining patients were considered to have pseudo-pseudohypoparathyroid (Pseudo-PHP) and were divided into two subtypes. One subtype had decreased Gsalpha and the second subtype had normal Gsalpha activity. The heterogeneous expression of Pseudo-PHP and thyrotropin resistance, which preceded parathyroid hormone resistance in 24% of the children, suggested that PHP might be a gradually evolving disease. GRF resistance was found in 4 out of 9 children investigated. The pedigree analysis showed PHP Ia had a dominant mode of inheritance with increased severity through generations. Pedigree analysis did not support a genomic imprinting hypothesis. Two children out of 9 had a chromosome 2 abnormality. This study confirms that Gsalpha activity is a significant marker in the diagnosis and classification of PHP.     

Pseudohypoparathyreoidismus und epigenetische Veränderungen des GNAS-Genlocus

The term pseudohypoparathyroidism (PHP) describes a heterogeneous group of related disorders characterized by end-organ resistance to parathyroid hormone (PTH). PHP is caused by deficiency of the α-subunit of stimulatory G proteins (Gsα), which is crucial for signal transduction of more than 1000 G protein-coupled receptors into the cell. PHP type Ia is caused by heterozygous, maternally inherited inactivating mutations involving those exons of the GNAS locus that encode Gsα. In addition, PHP Ia and Ic patients present with features of Albright hereditary osteodystrophy (AHO), which includes round face, short stature, brachymetacarpia, ectopic ossification, and mental retardation. Paternally inherited GNAS mutations lead to pseudo-PHP and are characterized by only some features of AHO in the absence of hormone resistance. PHP type Ib is caused by heterozygous, maternally inherited deletions up-stream of or within the GNAS locus that are associated with the loss of methylation at one or more maternally methylated regions within GNAS . Typically, these patients lack AHO features. This article provides an overview of the role of epigenetic factors for different PHP subtypes.     

Heterotopic ossifications in a mouse model of albright hereditary osteodystrophy

Albright hereditary osteodystrophy (AHO) is characterized by short stature, brachydactyly, and often heterotopic ossifications that are typically subcutaneous. Subcutaneous ossifications (SCO) cause considerable morbidity in AHO with no effective treatment. AHO is caused by heterozygous inactivating mutations in those GNAS exons encoding the α-subunit of the stimulatory G protein (Gα(s)). When inherited maternally, these mutations are associated with obesity, cognitive impairment, and resistance to certain hormones that mediate their actions through G protein-coupled receptors, a condition termed pseudohypoparathyroidism type 1a (PHP1a). When inherited paternally, GNAS mutations cause only AHO but not hormonal resistance, termed pseudopseudohypoparathyroidism (PPHP). Mice with targeted disruption of exon 1 of Gnas (Gnas(E1-/+)) replicate human PHP1a or PPHP phenotypically and hormonally. However, SCO have not yet been reported in Gnas(E1+/-) mice, at least not those that had been analyzed by us up to 3 months of age. Here we now show that Gnas(E1-/+) animals develop SCO over time. The ossified lesions increase in number and size and are uniformly detected in adult mice by one year of age. They are located in both the dermis, often in perifollicular areas, and the subcutis. These lesions are particularly prominent in skin prone to injury or pressure. The SCO comprise mature bone with evidence of mineral deposition and bone marrow elements. Superficial localization was confirmed by radiographic and computerized tomographic imaging. In situ hybridization of SCO lesions were positive for both osteonectin and osteopontin. Notably, the ossifications were much more extensive in males than females. Because Gnas(E1-/+) mice develop SCO features that are similar to those observed in AHO patients, these animals provide a model system suitable for investigating pathogenic mechanisms involved in SCO formation and for developing novel therapeutics for heterotopic bone formation. Moreover, these mice provide a model with which to investigate the regulatory mechanisms of bone formation.     

G protein defects in signal transduction

G proteins couple receptors for many hormones to effectors that regulate second messenger metabolism. Several endocrine disorders have been shown to be caused by either loss- or gain-of-function mutations in G proteins or G protein-coupled receptors. In pseudohypoparathyroidism type Ia (PHP Ia), there are generalized hormone resistance (parathyroid hormone [PTH], thyroid-stimulating hormone, gonadotropins) and associated abnormal physical features, Albright hereditary osteodystrophy. Subjects with PHP Ib are normal in appearance and show renal resistance to PTH. In McCune-Albright syndrome (MAS), subjects show autonomous endocrine hyperfunction associated with fibrous dysplasia of bone and skin hyperpigmentation. Germline loss-of-function mutations have been identified in the G(s)-alpha gene in PHP Ia, and recent evidence suggests that the G(s)-alpha gene is paternally imprinted in a tissue-specific manner. Abnormal imprinting of the G(s)-alpha gene may be the cause of PHP Ib. MAS, in contrast, is caused by gain-of-function missense mutations of the G(s)-alpha gene.     

A novel STX16 deletion in autosomal dominant pseudohypoparathyroidism type Ib redefines the boundaries of a cis-acting imprinting control element of GNAS

A unique heterozygous 3-kb microdeletion within STX16, a closely linked gene centromeric of GNAS, was previously identified in multiple unrelated kindreds as a cause of autosomal dominant pseudohypoparathyroidism type Ib (AD-PHP-Ib). We now report a novel heterozygous 4.4-kb microdeletion in a large kindred with AD-PHP-Ib. Affected individuals from this kindred share an epigenetic defect that is indistinguishable from that observed in patients with AD-PHP-Ib who carry the 3-kb microdeletion in the STX16 region (i.e., an isolated loss of methylation at GNAS exon A/B). The novel 4.4-kb microdeletion overlaps with the previously identified deletion by 1,286 bp and, similar to the latter deletion, removes several exons of STX16 (encoding syntaxin-16). Because these microdeletions lead to AD-PHP-Ib only after maternal transmission, we analyzed expression of this gene in lymphoblastoid cells of affected individuals with the 3-kb or the 4.4-kb microdeletion, an individual with a NESP55 deletion, and a healthy control. We found that STX16 mRNA was expressed in all cases from both parental alleles. Thus, STX16 is apparently not imprinted, and a loss-of-function mutation in one allele is therefore unlikely to be responsible for this disorder. Instead, the region of overlap between the two microdeletions likely harbors a cis-acting imprinting control element that is necessary for establishing and/or maintaining methylation at GNAS exon A/B, thus allowing normal G alpha(s) expression in the proximal renal tubules. In the presence of either of the two microdeletions, parathyroid hormone resistance appears to develop over time, as documented in an affected individual who was diagnosed at birth with the 4.4-kb deletion of STX16 and who had normal serum parathyroid hormone levels until the age of 21 mo.     

A Sporadic Case of Pseudohypoparathyroidism Type 1 and Idiopathic Primary Adrenal Insufficiency Associated with a Novel Mutation in the Gnas1 Gene

ObjectiveWe report an atypical association of primary adrenal insufficiency and pseudohypoparathyroidism (PHP) and a novel GNAS1 gene mutation in a Caucasian female who initially presented with adrenal crisis.MethodsA case report and literature review.ResultsA 37-year-old female presented with shock at 11 years of age, and investigations revealed primary adrenal insufficiency and pseudohypoparathyroidism (PHP). She had typical features of Albright hereditary osteodystrophy (AHO) and evidence of thyroid-stimulating hormone (TSH), growth-hormone-releasing hormone (GHRH), and gonadotrophin resistance fitting with the diagnosis of PHP type 1a/1c. She did not have a family history of any autoimmune disease or PHP. Her mother was phenotypically normal. Genomic DNA sequencing of those GNAS exons and adjacent intronic regions that encode the stimulatory guanine nucleotide-binding protein Gsαrevealed a novel heterozygous mutation in exon 11, c.857-858delCT.ConclusionThe association of primary adrenal insufficiency and PHP has not been reported in literature and may prove an area for further research. The novel mutation in this case adds to the spectrum of mutations associated with these disorders. (Endocr Pract. 2014;20:e202-e206)     

Albright's hereditary osteodystrophy associated with cerebellar pilocytic astrocytoma: coincidence or genetic relationship?

Albright's hereditary osteodystrophy (AHO) is a rare inherited disease characterized by skeletal abnormalities, short stature, and, in some cases, resistance to parathyroid hormone, resulting in pseudohypoparathyroidism (PHP). Heterozygous inactivating mutations of the GNAS1 gene are responsible for reduced activity of the alpha subunit of the Gs protein (G(Salpha)), a protein that mediates hormone signal transduction across cell membranes. G(salpha) is also known to have oncogenic potentials, leading to the development of human pituitary tumors and Leydig cell tumors. Here, we report the 1st case, a 3.5-year-old girl, with classic AHO phenotype and PHP type 1A associated with a cerebellar pilocytic astrocytoma. Coincidence or genetic relationships of both diseases are discussed according to molecular findings and current literature.     

Selective resistance to parathyroid hormone caused by a novel uncoupling mutation in the carboxyl terminus of G alpha(s). A cause of pseudohypoparathyroidism type Ib

G(s) is a heterotrimeric (alpha, beta, and gamma chains) G protein that couples heptahelical plasma membrane receptors to stimulation of adenylyl cyclase. Inactivation of one GNAS1 gene allele encoding the alpha chain of G(s) (G alpha(s)) causes pseudohypoparathyroidism type Ia. Affected subjects have resistance to parathyroid hormone (PTH) and other hormones that activate adenylyl cyclase plus somatic features termed Albright hereditary osteodystrophy. By contrast, subjects with pseudohypoparathyroidism type Ib have hormone resistance that is limited to PTH and lack Albright hereditary osteodystrophy. The molecular basis for pseudohypoparathyroidism type Ib is unknown. We analyzed the GNAS1 gene for mutations using polymerase chain reaction to amplify genomic DNA from three brothers with pseudohypoparathyroidism type Ib. We identified a novel heterozygous 3-base pair deletion causing loss of isoleucine 382 in the three affected boys and their clinically unaffected mother and maternal grandfather. This mutation was absent in other family members and 15 additional unrelated subjects with pseudohypoparathyroidism type Ib. To characterize the signaling properties of the mutant G alpha(s), we used site-directed mutagenesis to introduce the isoleucine 382 deletion into a wild type G alpha(s) cDNA, transfected HEK293 cells with either wild type or mutant G alpha(s) cDNA, plus cDNAs encoding heptahelical receptors for PTH, thyrotropic hormone, or luteinizing hormone, and we measured cAMP production in response to hormone stimulation. The mutant G alpha(s) protein was unable to interact with the receptor for PTH but showed normal coupling to the other coexpressed heptahelical receptors. These results provide evidence of selective uncoupling of the mutant G alpha(s) from PTH receptors and explain PTH-specific hormone resistance in these three brothers with pseudohypoparathyroidism type Ib. The absence of PTH resistance in the mother and maternal grandfather who carry the same mutation is consistent with current models of paternal imprinting of the GNAS1 gene.     

Clinical heterogeneity of pseudohypoparathyroidism: from hyper- to hypocalcemia

Pseudohypoparathyroidism (PHP) is a rare inherited syndrome characterized by parathyroid hormone (PTH) resistance and is frequently associated with Albright's hereditary osteodystrophy and resistance to other cAMP-mediated hormones. The usual neonatal presentation is mild primary hypothyroidism secondary to resistance to thyroid-stimulating hormone; hypocalcemia usually develops after age 3-5 years. This work describes the diversity in the clinical expression and course of PHP, with emphasis on calcium levels by age and treatment, in 8 children under long-term follow-up at our pediatric tertiary center. The calcium levels at presentation ranged from transient neonatal hypocalcemia to infantile hypercalcemia to childhood/adolescence hypocalcemia. Interestingly, relative hypocalciuria at diagnosis and during therapy, in the presence of renal PTH resistance, was the rule. These findings indicate that transient neonatal hypocalcemia associated with other clinical features or a family history of PHP may be a flag for clinicians to screen for PTH resistance later in life. In addition, PTH resistance may be missed by surveying calcium levels only; thus the PTH levels have to be checked as well. In addition, the recommendation for patients with hypoparathyroidism that strict low-normal calcium levels be maintained during therapy in order to prevent hypercalciuria is probably not applicable in PHP.     

Pseudohypoparathyroidism Type Ib Associated with Novel Duplications in the GNAS Locus

ContextPseudohypoparathyroidism type 1b (PHP-Ib) is characterized by renal resistance to PTH (and, sometimes, a mild resistance to TSH) and absence of any features of Albright''s hereditary osteodystrophy. Patients with PHP-Ib suffer of defects in the methylation pattern of the complex GNAS locus. PHP-Ib can be either sporadic or inherited in an autosomal dominant pattern. Whereas familial PHP-Ib is well characterized at the molecular level, the genetic cause of sporadic PHP-Ib cases remains elusive, although some molecular mechanisms have been associated with this subtype.ObjectiveThe aim of the study was to investigate the molecular and imprinting defects in the GNAS locus in two unrelated patients with PHP-Ib.DesignWe have analyzed the GNAS locus by direct sequencing, Methylation-Specific Multiplex Ligation-dependent Probe Amplification, microsatellites, Quantitative Multiplex PCR of Short Fluorescent fragments and array-Comparative Genomic Hybridization studies in order to characterize two unrelated families with clinical features of PHP-Ib.ResultsWe identified two duplications in the GNAS region in two patients with PHP-Ib: one of them, comprising ∼320 kb, occurred ‘de novo’ in the patient, whereas the other one, of ∼179 kb in length, was inherited from the maternal allele. In both cases, no other known genetic cause was observed.ConclusionIn this article, we describe the to-our-knowledge biggest duplications reported so far in the GNAS region. Both are associated to PHP-Ib, one of them occurring ‘de novo’ and the other one being maternally inherited.     

Pseudohypoparathyroidism type Ia: two new heterozygous frameshift mutations in exons 5 and 10 of the Gsα gene

Pseudohypoparathyroidism type Ia (PHP-Ia) is a hereditary disease characterized by resistance to PTH and other hormones that act via cAMP. Patients have deficient activity of Gs, the subunit of the G protein, which couples hormone receptors to stimulation of adenylate cyclase. We describe two new mutations discovered in two sporadic patients with PHP-Ia. Using genomic DNA, we have amplified exons 2–13 of the Gs gene (GNAS1) by PCR, and sequenced the resulting products. Both patients had Albright's hereditary osteodystrophy, resistance to multiple hormones, and deficient Gs activity. In the first patient, a deletion of a C in exon 5 at codon 115 was found. In the second patient, an insertion of a C in exon 10 at codon 267 was detected. Both these heterozygous mutations cause frameshift, and predict decreased production of Gs. This report adds two new Gs mutations to the known ten mutations recently described.     

Effect of calcium administration on renal responsiveness to parathyroid hormone in pseudohypoparathyroidism type I and II -- in comparison with normals, idiopathic and surgical hypoparathyroidism.

A 31-year-old man and a 12-year-old girl were diagnosed as pseudohypoparathyroidism (PHP) Type I because of a failure to respond to the administration of parathyroid hormone (PTH) with increased urinary excretion of phosphate and cyclic adenosine-3', 5'-monophosphate (cAMP). A 22-year-old woman was diagnosed as PHP Type II because there was no increase in the urinary excretion of phosphate despite of a marked increase in urinary cAMP excretion. With the combined calcium-PTH infusion or PTH infusion after vitamin D therapy, renal response was improved in these patients. Also dibutyryl adenosine-3'-5'-cyclic monophosphate (dbcAMP) infusion evoked an increased urinary phosphate excretion in all of the patients. The metabolic defect of our patients with PHP Type I may be caused not by a lack or defective form of PTH-sensitive receptor adenylate cyclase complex but rather by an abnormal conformation in the plasma membrane-associated receptor adenylate cyclase enzyme complex in kidney. In the patient with PHP Type II, as cAMP generation is intact, the metabolic defect might be related to a defect of calcium mobilization in renal tubular cells in response to PTH.