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 _s α) 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 _s α 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.

GNAS is a complex imprinted gene that uses multiple promoters to generate several gene products, including the G protein α-subunit (G_sα) that couples seven-transmembrane receptors to the cAMP-generating enzyme adenylyl cyclase. Somatic activating G_sα mutations, which alter key residues required for the GTPase turn-off reaction, are present in various endocrine tumors and fibrous dysplasia of bone, and in a more widespread distribution in patients with McCune- Albright syndrome. Heterozygous inactivating G_sα mutations lead to Albright hereditary osteodystrophy. G_sα is imprinted in a tissue-specific manner, being primarily expressed from the maternal allele in renal proximal tubules, thyroid, pituitary, and ovary. Maternally inherited mutations lead to Albright hereditary osteodystrophy (AHO) plus PTH, TSH, and gonadotropin resistance (pseudohypoparathyroidism type 1A), whereas paternally inherited mutations lead to AHO alone. Pseudohypoparathyroidism type 1B, in which patients develop PTH resistance without AHO, is almost always associated with a GNAS imprinting defect in which both alleles have a paternal-specific imprinting pattern on both parental alleles. Familial forms of the disease are associated with a mutation within a closely linked gene that deletes a region that is presumably required for establishing the maternal imprint, and therefore maternal inheritance of the mutation results in the GNAS imprinting defect. Imprinting of one differentially methylated region within GNAS is virtually always lost in pseudohypoparathyroidism type 1B, and this region is probably responsible for tissue-specific G_sα imprinting. Mouse knockout models show that G_sα and the alternative G_sα isoform XLαs that is expressed from the paternal GNAS allele may have opposite effects on energy metabolism in mice.

Familial multiple endocrine neoplasia type 1 (MEN-1) is characterized by tumors of the parathyroids, endocrine pancreas, and anterior pituitary. Since the gene associated with MEN-1, located on chromosome 11 (11q13), may normally inhibit tumor proliferation, tumors could arise from inactivation of one or both of the alleles. However, parathyroid tumors in patients with MEN-1 have been considered to result from polyclonal hyperplasia. Using genetic probes, we tested parathyroid tumors for a monoclonal component, represented by a loss of alleles at any of eight loci along chromosome 11.

Pseudohypoparathyroidism type IB (PHPIB) is characterized by renal resistance to parathyroid hormone (PTH) and the absence of other endocrine or physical abnormalities. Familial PHPIB has been mapped to 20q13, near GNAS1, which encodes G_sα, the G protein α-subunit required for receptor-stimulated cAMP generation. However, G_sα function is normal in blood cells from PHPIB patients, ruling out mutations within the G_sα coding region. In mice G_sα is expressed only from the maternal allele in renal proximal tubules (the site of PTH action) but is biallelically expressed in most other tissues. Studies in patients with Albright hereditary osteodystrophy suggest a similar G_sα imprinting pattern in humans. Here we identify a region upstream of the G_sα promoter that is normally methylated on the maternal allele and unmethylated on the paternal allele, but that is unmethylated on both alleles in all 13 PHPIB patients studied. Within this region is an alternative promoter and first exon (exon 1A), generating transcripts that are normally expressed only from the paternal allele, but that are biallelically expressed in PHPIB patients. Therefore, PHPIB is associated with a paternal-specific imprinting pattern of the exon 1A region on both alleles, which may lead to decreased G_sα expression in renal proximal tubules. We propose that loss of exon 1A imprinting is the cause of PHPIB.

Osteoblasts play an increasingly recognized role in supporting hematopoietic development and recently have been implicated in the regulation of B lymphopoiesis. Here we demonstrate that the heterotrimeric G protein α subunit G _s α is required in cells of the osteoblast lineage for normal postnatal B lymphocyte production. Deletion of G _s α early in the osteoblast lineage results in a 59% decrease in the percentage of B cell precursors in the bone marrow. Analysis of peripheral blood from mutant mice revealed a 67% decrease in the number of circulating B lymphocytes by 10 days of age. Strikingly, other mature hematopoietic lineages are not decreased significantly. Mice lacking G _s α in cells of the osteoblast lineage exhibit a reduction in pro-B and pre-B cells. Furthermore, interleukin (IL)-7 expression is attenuated in G _s α-deficient osteoblasts, and exogenous IL-7 is able to restore B cell precursor populations in the bone marrow of mutant mice. Finally, the defect in B lymphopoiesis can be rescued by transplantation into a WT microenvironment. These findings confirm that osteoblasts are an important component of the B lymphocyte niche and demonstrate in vivo that G _s α-dependent signaling pathways in cells of the osteoblast lineage extrinsically regulate bone marrow B lymphopoiesis, at least partially in an IL-7-dependent manner.