The stimulatory G protein alpha-subunit Gs alpha is imprinted in human thyroid glands: implications for thyroid function in pseudohypoparathyroidism types 1A and 1B

The stimulatory G protein alpha-subunit G(s)alpha couples receptors to adenylyl cyclase and is required for hormone-stimulated cAMP generation. In Albright hereditary osteodystrophy, heterozygous G(s)alpha null mutations only lead to PTH, TSH, and gonadotropin resistance when inherited maternally [pseudohypoparathyroidism type 1A; (PHP1A)]. Maternal-specific expression of G(s)alpha in specific hormone targets could explain this observation. Using hot-stop PCR analysis on total RNA from six normal human thyroid specimens, we showed that the majority of the G(s)alpha mRNA (72 +/- 3%) was derived from the maternal allele. This is consistent with the presence of TSH resistance in patients with maternal G(s)alpha null mutations (PHP1A) and the absence of TSH resistance in patients with paternal G(s)alpha mutations (pseudopseudohypoparathyroidism). Patients with PTH resistance in the absence of Albright hereditary osteodystrophy (PHP1B) have an imprinting defect of the G(s)alpha gene resulting in both alleles having a paternal epigenotype, which would lead to a more moderate level of thyroid-specific G(s)alpha deficiency. We found evidence of borderline TSH resistance in 10 of 22 PHP1B patients. This study provides further evidence for tissue-specific imprinting of G(s)alpha in humans and provides a potential mechanism for mild to moderate TSH resistance in PHP1A and borderline resistance in some patients with PHP1B.     

A mouse model of albright hereditary osteodystrophy generated by targeted disruption of exon 1 of the Gnas gene

Albright hereditary osteodystrophy is caused by heterozygous inactivating mutations in GNAS, a gene that encodes not only the alpha-chain of Gs (Galphas), but also NESP55 and XLalphas through use of alternative first exons. Patients with GNAS mutations on maternally inherited alleles are resistant to multiple hormones such as PTH, TSH, LH/FSH, GHRH, and glucagon, whose receptors are coupled to Gs. This variant of Albright hereditary osteodystrophy is termed pseudohypoparathyroidism type 1a and is due to presumed tissue-specific paternal imprinting of Galphas. Previous studies have shown that mice heterozygous for a targeted disruption of exon 2 of Gnas, the murine homolog of GNAS, showed unique phenotypes dependent on the parent of origin of the mutated allele. However, hormone resistance occurred only when the disrupted gene was maternally inherited. Because disruption of exon 2 is predicted to inactivate Galphas as well as NESP55 and XLalphas, we created transgenic mice with disruption of exon 1 to investigate the effects of isolated loss of Galphas. Heterozygous mice that inherited the disruption maternally (-m/+) exhibited PTH and TSH resistance, whereas those with paternal inheritance (+/-p) had normal hormone responsiveness. Heterozygous mice were shorter and, when the disrupted allele was inherited maternally, weighed more than wild-type littermates. Galphas protein and mRNA expression was consistent with paternal imprinting in the renal cortex and thyroid, but there was no imprinting in renal medulla, heart, or adipose. These findings confirm the tissue-specific paternal imprinting of GNAS and demonstrate that Galphas deficiency alone is sufficient to account for the hormone resistance of pseudohypoparathyroidism type 1a.     

GNAS1 lesions in pseudohypoparathyroidism Ia and Ic: genotype phenotype relationship and evidence of the maternal transmission of the hormonal resistance.

We conducted clinical and biological studies including screening for mutations in the gene encoding the alpha subunit of G(s) (GNAS1) in 30 subjects (21 unrelated families) with Albright's hereditary osteodystrophy (AHO), pseudohypoparathyroidism (PHP); and decreased erythrocyte G(s) activity (PHP-Ia; n = 19); AHO and decreased erythrocyte G(s) activity (isolated AHO; n = 10); or AHO, hormonal resistance, and normal erythrocyte G(s) activity (PHP-Ic; n = 1). A heterozygous GNAS1 gene lesion was found in 14 of 17 PHP-Ia index cases (82%), including 11 new mutations and a mutational hot-spot involving codons 189-190 (21%). These lesions lead to a truncated protein in all but three cases with missense mutations R280K, V159M, and D156N. In the patient diagnosed with PHP-Ic, G(s)alpha protein was shortened by just four amino acids, a finding consistent with the conservation of G(s) activity in erythrocytes and the loss of receptor contact. No GNAS1 lesions were found in individuals with isolated AHO that were not relatives to PHP-Ia patients (n = 5). Intrafamilial segregation analyses of the mutated GNAS1 allele in nine PHP-Ia patients established that the mutation had either occurred de novo on the maternal allele (n = 4) or had been transmitted by a mother with a mild phenotype (n = 5). This finding is consistent with an imprinting of GNAS1 playing a role in the clinical phenotype of loss of function mutations and with a functional maternal GNAS1 allele having a predominant role in preventing the hormonal resistance of PHP-Ia.     

G protein mutations in endocrine diseases.

This review summarizes the pathogenetic role of naturally occurring mutations of G protein genes in endocrine diseases. Although in vitro mutagenesis and transfection assays indicate that several G proteins have mitogenic potential, to date only two G proteins have been identified which harbor naturally occurring mutations, Gsalpha, the activator of adenylyl cyclase and Gi2alpha, which is involved in several functions, including adenylyl cyclase inhibition and ion channel modulation. The gene encoding Gsalpha (GNAS1) may be altered by loss or gain of function mutations. Indeed, heterozygous inactivating germ line mutations in this gene cause pseudohypoparathyroidism type Ia, in which physical features of Albright hereditary osteodystrophy (AHO) are associated with resistance to several hormones, i.e. PTH, TSH and gonadotropins, that activate Gs-coupled receptors or pseudopseudohypoparathyroidism in which AHO is the only clinical manifestation. Evidence suggests that the variable and tissue-specific hormone resistance observed in PHP Ia may result from tissue-specific imprinting of the GNAS1 gene, although the Gsalpha knockout model only in part reproduces the human AHO phenotype. Activating somatic Gsalpha mutations leading to cell proliferation have been identified in endocrine tumors constituted by cells in which cAMP is a mitogenic signal, i.e. GH-secreting pituitary adenomas, hyperfunctioning thyroid adenomas and Leydig cell tumors. When the same mutations occur very early in embryogenesis they cause McCune-Albright syndrome. Although these mutations would in principle confer growth advantage, studies failed to detect differences in the clinical and hormonal phenotypes, suggesting the existence of mechanisms able to counteract the activation of the cAMP pathway. Activating mutations of Gi2alpha have been identified in a subset of ovarian, adrenal and pituitary tumors, but their prevalence and significance are still controversial. Finally, although Galpha subunits are the only components of the heterotrimeric GTP binding proteins which harbor known mutations, beta/gamma subunits should be considered possible targets of genetic alterations as suggested by the frequent presence of beta3 subunit variants in patients with essential hypertension.     

The Role of Genomic Imprinting of Gsα in the Pathogenesis of Albright Hereditary Osteodystrophy

Albright hereditary osteodystrophy (AHO) is caused by heterozygous inactivating mutations of the gene encoding the α-subunit of the G protein G_s. The G_sα gene is a complex gene that uses various alternative promoters and produces various protein products. Recently, it has been shown that this gene is imprinted in a tissue-specific manner. The role of tissue-specific imprinting of G_sα in the pathogenesis of AHO is discussed.     

Pseudohypoparathyroidism type Ia. Albright hereditary osteodystrophy: a model for research on G protein-coupled receptors and genomic imprinting

Pseudohypoparathyroidism type Ia (PHP Ia) is a hereditary endocrine disorder, characterised by resistance to parathyroid hormone (PTH), causing disturbance of calcium homeostasis, and to several other polypeptide hormones. Patients with PHP Ia exhibit a complex of somatic abnormalities, termed Albright hereditary osteodystrophy (AHO). Treatment with vitamin D derivatives alleviates symptoms of hypocalcemia and may prevent bone demineralisation. PTH, like many polypeptide hormones, exerts its effects via a G protein-coupled cell surface receptor. PHP Ia is caused by a heterozygous, inactivating mutation in the gene for the alpha-subunit of the Gs protein, which disrupts Gs-protein-coupled signal transduction pathways. Several mutations have been described. When the mutation is inherited from the mother, the offspring will develop PHP Ia, i.e., both hormonal resistance and somatic abnormalities. When the mutation is derived from the father, children will have normal hormone responses while exhibiting the somatic features of AHO; this form of the disorder is called pseudopseudohypoparathyroidism (PPHP). A combination of tissue-specific genomic imprinting and haploinsufficiency may explain the occurrence of PPHP, and the fact that not all Gs-mediated polypeptide hormone actions are affected equally. PHP may therefore serve as a model in studying the pleiotropic consequences of impaired Gs-mediated signal transduction.     

Analysis of the GNAS1 gene in Albright's hereditary osteodystrophy

Albright's hereditary osteodystrophy (AHO) is characterized by phenotypic signs that typically include brachydactyly and sc calcifications occurring with or without hormone resistance toward PTH or other hormones such as thyroid hormone or gonadotropins. Different inactivating mutations of the gene GNAS1 encoding Gsalpha lead to a reduced Gsalpha protein activity in patients with AHO and pseudohypoparathyroidism type Ia or without resistance to PTH (pseudopseudohypoparathyroidism). We investigated 29 unrelated patients with AHO and pseudohypoparathyroidism type Ia or pseudopseudohypoparathyroidism and their affected family members performing functional and molecular genetic analysis of Gsalpha. In vitro determination of Gsalpha protein activity in erythrocyte membranes was followed by the investigation of the whole coding region of the GNAS1 gene using PCR, nonisotopic single strand conformation analysis, and direct sequencing of the PCR products. All patients showed a reduced Gsalpha protein activity (mean 59% compared with healthy controls). In 21/29 (72%) patients, 15 different mutations in GNAS1 including 11 novel mutations were detected. In addition we add five unrelated patients with a previously described 4 bp deletion in exon 7 (Delta GACT, codon 189/190), confirming the presence of a hot spot for loss of function mutations in GNAS1. In eight patients, no molecular abnormality was found in the GNAS1 gene despite a functional defect of Gsalpha. We conclude that biochemical and molecular analysis of Gsalpha and its gene GNAS1 can be valuable tools to confirm the diagnosis of AHO. However, in some patients with reduced activity of Gsalpha, the molecular defect cannot be detected in the exons encoding the common form of Gsalpha.     

Identification of two novel deletion mutations within the Gs alpha gene (GNAS1) in Albright hereditary osteodystrophy.

Albright hereditary osteodystrophy (AHO) is a genetic disorder characterized by short stature, skeletal defects, and obesity. Within AHO kindreds, some affected family members have only the somatic features of AHO [pseudopseudohypoparathyroidism (PPHP)], whereas others have these features in association with resistance to multiple hormones that stimulate adenylyl cyclase within their target tissues [pseudohypoparathyroidism type Ia (PHP Ia)]. Affected members of most AHO kindreds (both those with PPHP and those with PHP Ia) have a partial deficiency of Gs alpha, the alpha-subunit of the G protein that couples receptors to adenylyl cyclase stimulation, and in a number of cases heterozygous loss of function mutations within the Gs alpha gene (GNAS1) have been identified. Using PCR with the attachment of a high melting domain (GC-clamp) and temperature gradient gel electrophoresis, two novel heterozygous frameshift mutations within GNAS1 were found in two AHO kindreds. In one kindred all affected members (both PHP Ia and PPHP) had a heterozygous 2-bp deletion in exon 8, whereas in the second kindred a heterozygous 2-bp deletion in exon 4 was identified in all affected members examined. In both cases the frameshift encoded a premature termination codon several codons downstream of the deletion. In the latter kindred affected members were previously shown to have decreased levels of GNAS1 messenger ribonucleic acid expression. These results further underscore the genetic heterogeneity of AHO and provides further evidence that PHP Ia and PPHP are two clinical presentations of a common genetic defect. Serial measurements of thyroid function in members of kindred 1 indicate that TSH resistance progresses with age and becomes more evident after the first year of life.     

Body mass index differences in pseudohypoparathyroidism type 1a versus pseudopseudohypoparathyroidism may implicate paternal imprinting of Galpha(s) in the development of human obesity

CONTEXT: Obesity is a prominent feature of Albright hereditary osteodystrophy (AHO), a disorder caused by heterozygous GNAS mutations that disrupt the stimulatory G protein alpha-subunit Galpha(s). Because Galpha(s) is paternally imprinted in certain hormone target tissues, maternal inheritance of AHO leads to multihormone resistance [pseudohypoparathyroidism type 1a (PHP1a)], whereas paternal inheritance leads to AHO alone [pseudopseudohypoparathyroidism (pseudoPHP)]. Classically, the obesity in AHO is described as occurring similarly in both conditions. SETTING: This observational study was conducted at the General Clinical Research Center, Johns Hopkins University School of Medicine; National Institutes of Health. PATIENTS: Fifty-three patients with AHO (40 with PHP1a and 13 with pseudoPHP) and two with progressive osseous heteroplasia were studied. MAIN OUTCOME MEASURES: Main outcome measures were weight and height sd score (SDS), body mass index (BMI) percentiles, and BMI z-scores. RESULTS: Patients with PHP1a had significantly greater mean weight SDS, BMI percentages, and BMI z-scores compared with patients with pseudoPHP. These differences in BMI were secondary to adipose content based on dual energy x-ray absorptiometry analysis. The mean BMI z-score +/- sem for PHP1a was 2.31 +/- 0.18 compared with 0.65 +/- 0.31 in pseudoPHP (P = 0.000032). Twenty-five of 40 (62.5%) patients with PHP1a had mean BMI z-scores greater than two SDS above the mean, whereas no patients with pseudoPHP had BMI z-scores in this range. CONCLUSIONS: Although the AHO phenotype for PHP1a and pseudoPHP has been thought to be similar, we have found that obesity is a more prominent feature in PHP1a than in pseudoPHP and that severe obesity is characteristic of PHP1a specifically. These findings may implicate paternal imprinting of Galpha(s) in the development of human obesity.     

A maternal epimutation of GNAS leads to Albright osteodystrophy and parathyroid hormone resistance

CONTEXT: Pseudohypoparathyroidism (PHP) type Ia is a rare maternally transmitted disease due to maternal loss-of-function mutations of GNAS, the gene encoding Galphas, the alpha-stimulatory subunit of the G protein. Affected individuals display hormonal resistance (mainly PTH and TSH resistance) and Albright hereditary osteodystrophy. PHP type Ib (PHP-Ib), usually defined by isolated renal resistance to PTH and sometimes mild TSH resistance, is due to a maternal loss of GNAS exon A/B methylation, leading to decreased Galphas expression in specific tissues. OBJECTIVE AND RESULTS: We report a girl with obvious Albright osteodystrophy features, PTH resistance, normal Galphas bioactivity in red blood cells, yet no loss-of-function mutation in the GNAS coding sequence (exons 1-13). The methylation analysis of the four GNAS differentially methylated regions, i.e. NESP, AS, XL, and A/B, revealed broad methylation changes at all differentially methylated regions, including GNAS exon A/B, leading to a paternal epigenotype on both alleles. CONCLUSIONS: This observation suggests that: 1) the decreased expression of Galphas due to GNAS epimutations is not restricted to the renal tubule but may affect nonimprinted tissues like bone; 2) PHP-Ib is a heterogeneous disorder that should lead to studying GNAS epigenotype in patients with PHP and no mutation in GNAS exons 1-13, regardless of their physical features.     

Variable and tissue-specific hormone resistance in heterotrimeric Gs protein α-subunit (Gsα) knockout mice is due to tissue-specific imprinting of the Gsα gene

Albright hereditary osteodystrophy (AHO), an autosomal dominant disorder characterized by short stature, obesity, and skeletal defects, is associated with heterozygous inactivating mutations of GNAS1, the gene encoding the heterotrimeric G protein α-subunit (G_sα) that couples multiple receptors to the stimulation of adenylyl cyclase. It has remained unclear why only some AHO patients present with multihormone resistance and why AHO patients demonstrate resistance to some hormones [e.g., parathyroid hormone (PTH)] but not to others (e.g., vasopressin), even though all activate adenylyl cyclase. We generated mice with a null allele of the mouse homolog Gnas. Homozygous G_s deficiency is embryonically lethal. Heterozygotes with maternal (m−/+) and paternal (+/p−) inheritance of the Gnas null allele have distinct phenotypes, suggesting that Gnas is an imprinted gene. PTH resistance is present in m−/+, but not +/p−, mice. G_sα expression in the renal cortex (the site of PTH action) is markedly reduced in m−/+ but not in +/p− mice, demonstrating that the Gnas paternal allele is imprinted in this tissue. Gnas is also imprinted in brown and white adipose tissue. The maximal physiological response to vasopressin (urinary concentrating ability) is normal in both m−/+ and +/p− mice and Gnas is not imprinted in the renal inner medulla (the site of vasopressin action). Tissue-specific imprinting of Gnas is likely the mechanism for variable and tissue-specific hormone resistance in these mice and a similar mechanism might explain the variable phenotype in AHO.     

Phenotypic and molecular genetic aspects of pseudohypoparathyroidism type Ib in a Greek kindred: evidence for enhanced uric acid excretion due to parathyroid hormone resistance

The predominant feature of pseudohypoparathyroidism (PHP) is renal resistance to PTH. Pseudohypoparathyroidism type Ia (PHP-Ia) is caused by maternally inherited heterozygous mutations in the GNAS exons encoding the alpha-subunit of the stimulatory G protein (Gsalpha). Besides PTH resistance, PHP-Ia patients have Albright's hereditary osteodystrophy and often display resistance to additional hormones. Patients with PHP-Ib lack features of Albright's hereditary osteodystrophy, and PTH resistance is associated with loss of methylation at the maternal GNAS exon A/B. Most individuals with the autosomal dominant form of PHP-Ib have a 3-kb microdeletion within STX16 approximately 220 kb upstream of exon A/B. Here we report on the clinical and genetic aspects of a Greek PHP-Ib kindred with four affected members and three obligate carriers, who had the 3-kb deletion within STX16. Symptomatic hypocalcemia was present only in the proband, but PTH was elevated in all members who had inherited the 3-kb deletion maternally. In all affected family members, urinary phosphate excretion was normal, but 1,25-dihydroxyvitamin D levels were diminished. These findings confirm previous data regarding patient to patient variation in disease severity for autosomal dominant PHP-Ib. Furthermore, affected individuals displayed hypouricemia with increased fractional excretion of uric acid, suggesting possible involvement of PTH in the renal handling of this metabolite.     

Pseudohypoparathyroidism type Ib in 2015

The term pseudohypoparathryoidism (PHP) refers to a group of rare genetic and epigenetic disorders characterized by resistance to the action of parathyroid hormone (PTH) that activates cAMP signaling in target cells. Together with pseudohypoparathyroidism, Albright hereditary osteodystrophy (AHO) and progressive osseous heteroplasia (POH) represent rare, related and deeply impairing disorders encompassing heterogeneous features, such as brachydactyly, ectopic ossifications, short stature, mental retardation and endocrine deficiencies due to resistance to the action of different hormones. The two main subtypes, PHP-Ia and PHP-Ib, are caused by mutations in GNAS exons 1-13 and methylation defects in the imprinted GNAS cluster respectively, while mutations in the PRKAR1A and PDE4D genes (also involved in mediating cAMP signalling) have been demonstrated in patients with acrodysostosis, a disease of bone formation with characteristics similar to AHO. The molecular overlap among these disorders indicates the need for different classification models and seriously alters our understanding of the mechanisms through which GNAS defects, together with the new recently described defects involving other components of the cAMP signalling cascade, cause AHO-related disorders.     

Genetic analysis and evaluation of resistance to thyrotropin and growth hormone-releasing hormone in pseudohypoparathyroidism type Ib

CONTEXT: Pseudohypoparathyroidism (PHP) types Ia and Ib, are caused by mutations in GNAS exons 1-13 and GNAS methylation defects, respectively. PHP-Ia patients show Albright hereditary osteodystrophy (AHO) and resistance toward PTH and additional hormones, whereas PHP-Ib patients do not have AHO and hormone resistance is limited to PTH and, as reported in one paper, TSH. No study addressed the question of GH deficiency in PHP-Ib patients. OBJECTIVES: The objective of the study was to screen patients with clinically diagnosed PHP-Ib for genetic defects and investigate the presence of resistance to TSH and GHRH. PATIENTS/METHODS: We investigated GNAS differential methylation and STX16 microdeletions in genomic DNA from 10 patients with clinical diagnosis of sporadic PHP-Ib, i.e. PTH resistance without AHO. Resistance to GHRH was assessed by GH response to GHRH plus arginine. Thyroid function and ultrasonography were also evaluated. RESULTS: Molecular analysis showed GNAS cluster imprinting defects in all PHP-Ib patients and the first de novo STX16 deletion in one apparently sporadic patient. Subclinical or clinical hypothyroidism due to resistance to TSH was present in nine of 10 patients, whereas a preserved GH response to a GHRH plus arginine test was present in all patients, with one exception. CONCLUSIONS: We report the first molecular analysis of Italian patients with PHP-Ib. Clinical investigation shows that, like PHP-Ia patients, PHP-Ib patients are resistant to TSH, whereas they maintain a normal responsiveness to GHRH, at variance with PHP-Ia patients. These data provide new information on this rare disease and emphasize the clinical heterogeneity of genetic defects within GNAS.     

Preimplantation genetic diagnosis for severe albright hereditary osteodystrophy

CONTEXT: Preimplantation genetic diagnosis (PGD) enables the selection of embryos without mutations for implantation and has not been described to our knowledge for mutations in GNAS. Phocomelia in a patient with Albright hereditary osteodystrophy (AHO) has also not been previously described. OBJECTIVE: The aim of this study was to identify a GNAS mutation in a patient with a severe form of AHO and pseudohypoparathyroidism type 1a with phocomelia and to perform PGD on embryos derived by in vitro fertilization to deliver an unaffected infant. DESIGN: A proband and his family are described clinically, the GNAS gene was sequenced to identify a novel mutation in the proband, and PGD was performed on embryos. SETTING: The setting was in a tertiary-care hospital. PATIENTS: The patients were from a single family in which the proband has a severe form of AHO. INTERVENTIONS: Interventions were PGD and in vitro fertilization. MAIN OUTCOME MEASURES: The main outcome measures were the clinical phenotypes and GNAS gene sequences of the proband, embryos, and family members. RESULTS: After PGD, three genotypically normal embryos were transferred back to the mother. Pregnancy ensued, and a healthy male infant was delivered at 36.5 wk gestation. The GNAS genes in the baby were confirmed as wild-type, and the infant is free of any signs of AHO. CONCLUSIONS: We describe herein a proband with AHO and severe skeletal deformities (including phocomelia) related to a novel GNAS mutation and the delivery of a male infant with homozygous normal GNAS genotype after PGD.