6.16 Molecular and clinical characteristics of the McCune–Albright syndrome

Heterotrimeric guanine nucleotide-binding proteins (G proteins) couple extracellular receptor proteins to intracellular effector enzymes and ion channels. The observation that alterations in G protein-coupled signalling pathways can impact cellular function and proliferation, and cause human disease, has stimulated investigation into the molecular and pharmacological regulation of G protein expression and action. The most well characterized models for altered G protein expression defects have been based on naturally occurring mutations in GNAS, a complex gene at 20q13 which encodes the α subunit of Gs, the G protein that stimulates adenylyl cyclase. Somatic mutations in GNAS (OMIM 139320) that activate Gα_s are present in a subset of endocrine tumours and in patients with the McCune–Albright syndrome (OMIM 174800), a sporadic disorder characterized by increased hormone production and/or cellular proliferation of many tissues. By contrast, germline mutations of the GNAS gene that decrease expression or function of Gα_s are present in subjects with Albright’s hereditary osteodystrophy (AHO), a heritable disorder associated with a constellation of developmental defects and, in many patients, reduced responsiveness to multiple hormones that signal through receptors that require Gα_s to activate adenylyl cyclase EC 4.6.1.1 (i.e. pseudohypoparathyroidism type 1a (OMIM 103580)). McCune–Albright syndrome (MAS) and AHO represent contrasting gain of function and loss of function mutations in the GNAS gene, respectively. Clinical and biochemical analyses of subjects with these syndromes have extended our understanding of the developmental and functional consequences of dysfunctional G protein action, and have provided unexpected insights into the importance of cAMP as a regulator of the growth and/or function of many tissues. This chapter will focus on the clinical implications of activating mutations of GNAS as the basis for MAS.

G proteins share a common heterotrimeric structure, consisting of an α subunit and a tightly coupled βγ dimer. The α subunit interacts with detector and effector molecules, binds GTP, and possesses intrinsic GTPase activity (1). There are 16 genes in mammals that encode some 20 different α chains. The α subunits associate with a smaller group of β (at least five) and γ (more than 12) subunits (2). Combinatorial specificity in the associations between various G protein subunits provides the potential for enormous diversity, and may allow distinct heterotrimers to interact selectively with only a limited number of G protein-coupled receptors and effector proteins.

G protein-coupled signalling is regulated by a mechanism in which the binding and hydrolysis of GTP acts a molecular timing switch (Fig. 6.16.1). In the basal (inactive) state, G proteins exist in the heterotrimeric form with GDP bound to the α chain. The interaction of a ligand-bound receptor with a G protein facilitates the release of tightly bound GDP and the subsequent binding of cytosolic GTP. The binding of GTP to the α chain induces conformational changes that facilitate the dissociation of the α-GTP chain from the βγ dimer and the receptor. The free α-GTP chain assumes an active conformation in which a new surface is formed which enables the α chain to interact with target enzymes and ion channels with 20- to 100-fold higher affinity than in the GDP bound state. The βγ dimers also participate in downstream signalling events through interaction with an ever-widening array of targets, including certain forms of adenylyl cyclase and phospholipase C, potassium channels, and G protein-coupled receptor kinases.

G protein signalling is terminated by the hydrolysis of α-GTP to α-GDP by an intrinsic GTPase. The GTPase reaction is a high-energy transition state which requires association of the γ-phosphorus atom with the oxygen of a water molecule. To catalyse this reaction, the γ-phosphate of GTP must be stabilized so that a straight line, perpendicular to the plane of the γ-phosphate, connects the water, the γ-phosphorus, and the oxygen molecule leaving the β-phosphate. In Gα_s amino acids arginine²⁰¹ and glutamine²²⁷ function as ‘fingers’ to position the γ-phosphate of GTP. With hydrolysis of GTP to GDP, the α-GDP chain reassociates with the βγ dimer and the heterotrimeric G protein is capable of participating in another cycle of receptor-activated signalling (Fig. 6.16.1).

The GTPase of the Gα chain is a molecular timer that controls the duration, and thereby the intensity, of the signalling event. Different G protein α have distinctive rates of GTP hydrolysis, and changes in GTPase activity can have profound consequences on signalling. Several factors, termed ‘GTPase activating proteins’ (GAPs) (3), can interact directly with specific α chains to accelerate the slow intrinsic rate of GTP hydrolysis. One important class of GAPs is represented by the evolutionarily conserved superfamily of proteins, termed ‘regulators of G protein signalling’, that can stimulate a 40-fold increase in the catalytic rate of GTP hydrolysis, and thus can markedly accelerate the termination of G protein signalling. On the other hand, inhibition of intrinsic GTPase by modification or replacement of key amino acid residues (e.g. arginine²⁰¹ or glutamine²²⁷ in Gα_s) can delay termination of the signal transduction process, and cause persistent and excessive signalling. For example, exotoxins secreted by Vibrio cholerae and some strains of E. coli catalyse the addition of an ADP-ribose moiety to the side chain of arginine²⁰¹ in Gα_s. This covalent modification markedly reduces GTP hydrolysis and maintains Gα_s in its active GTP-bound form, thus resulting in persistent stimulation of adenylyl cyclase (4). The subsequent accumulation of cAMP in intestinal epithelial cells stimulates secretion of salt and water into the intestine and produces, in part, the watery diarrhoea associated with cholera.

Activity of adenylyl cyclase is under dual regulatory control through receptors that interact with either G_s to stimulate adenylyl cyclase or with G_i to inhibit adenylyl cyclase. Increased intracellular cAMP stimulates proliferation of many cell types, and can increase synthesis and secretion of endogenous hormones and neurotransmitters. Both germline and somatic mutations in GNAS that lead to a gain of function in Gα_s produce constitutive (i.e. hormone independent) activation of adenylyl cyclase (5, 6). Vallar, et al. (7) initially described a subset of human growth hormone-secreting pituitary tumours in which basal adenylyl cyclase activity in vitro was very high and failed to increase further with addition of growth hormone releasing hormone. Subsequent studies showed that these somatotropic tumours contained unusual forms of Gα_s that lacked GTPase activity. Loss of GTPase results from somatic mutations in GNAS that replace either arginine²⁰¹ or glutamine²²⁷ and thereby convert GNAS into the gsp oncogene. Arginine²⁰¹ corresponds to the site of choleragen modification of Gα_s (described above), whereas glutamine²²⁷ in Gα_s corresponds to the cognate amino acid Gln⁶¹ in the low-molecular-weight GTP-binding protein p21^ras. Naturally occurring Gln⁶¹ mutations convert p21^ras into an oncogene that plays a role in the development of a variety of human tumours (8). Replacement of either arginine²⁰¹ or glutamine²²⁷ in Gα_s enables the protein to remain in the active, GTP-bound state, and the consequent increase in cAMP leads to cellular proliferation and excessive hormone secretion (9, 10). Such activating mutations occur in approximately 40% of somatotropic tumours (Box 6.16.1). In addition to growth hormone-secreting pituitary tumours, gsp mutations are also present in a small number of ACTH-secreting pituitary tumours (11, 12), a subset of thyroid neoplasms, and testicular and ovarian stromal Leydig tumours (13), but are rare in other endocrine tumours (Box 6.16.1). Moreover, gsp mutations have been described in ovarian cysts that cause isosexual gonadotropin-independent precocious puberty (14, 15), in intramuscular myxomas (16), and in isolated fibrous dysplasia of the bone (17).

In 1937, McCune and Bruch (18) and Albright and associates (19) independently described a sporadic syndrome characterized by the clinical triad of polyostotic fibrous dysplasia, café-au-lait skin lesions, and endocrine hyperfunction, now known as McCune–Albright syndrome (MAS) (Fig. 6.16.2). Despite excessive activity of endocrine tissues, serum levels of the relevant regulatory or tropic hormones were either normal or decreased, suggesting autonomous function. Based on the observation that the cutaneous hyperpigmentation in MAS typically follows the developmental lines of Blaschko, Happle proposed that the underlying genetic abnormality might be a dominantly acting somatic mutation that occurs early in development, leading to a mosaic pattern of distribution of mutant cells (20). Similarly, a lack of documented heritability of MAS has been interpreted as evidence that germline transmission of the mutation would be lethal (20).

The molecular basis for MAS is a somatic mutation in exon 8 of GNAS which replaces the residue arginine at position 201, generally by histidine or cysteine (21, 22) but occasionally by serine, glycine, or leucine (23–29). Although missense mutations that replace the nearby glutamine at position 227 have been identified in solitary endocrine tumours, they have not been described in patients with MAS.

Consistent with a postzygotic somatic mutation, cells containing the gsp mutation are not present in all tissues of patients with MAS. Rather, cells containing a mutant GNAS gene are distributed in a mosaic pattern, with the greatest number of gsp-containing cells present in the most abnormal areas of affected tissues (Fig. 6.16.3) (21, 22, 24, 30, 31). In some cases, gsp alleles may be present in only some cell types within tissues that are derived from different embryological precursors. For example, a 3-year-old male MAS patient with macro-orchidism but no precocious puberty was reported to have an Arg201His gsp allele present only in Sertoli cells, resulting in isolated Sertoli cell hyperfunction, evidenced by increased AMH expression and cell hyperplasia leading to prepubertal macro-orchidism. There were no signs of Leydig cell activation, and no evidence of excess androgen action (32, 33). The different early embryologic origin of precursors contributing to Sertoli and Leydig cell lineages may underlie the differential distribution of the mutated GNAS gene.

The variable involvement of different tissues in patients with MAS, as well as the clinical heterogeneity among affected patients, is assumed to be a result of several unique features. First, the number of tissues in which the gsp is present, and the proportion and distribution of affected cells in a tissue, will be determined by the timing of the mutational event. Thus, mutations that arise early in embryogenesis are likely to affect several cell lineages and produce a more severe phenotype than mutational events that occur later. For example, acquisition of a gsp mutation months or even years after birth could explain the development of a solitary endocrine tumour or a single fibrous dysplasia lesion in some patients.

Second, epigenetic and/or microenvironmental factors that regulate GNAS expression can influence the MAS phenotype. For example, stochastic effects, such as allelic imbalance, may favour expression of the mutant allele in some tissues (34), thus exaggerating the effect of a gsp mutation. Even more importantly, tissue-specific imprinting of GNAS can exert a discrete effect on expression of gsp alleles. GNAS transcripts that encode Gα_s are preferentially expressed from the maternal allele in some cells (e.g. renal proximal tubule cells, thyroid follicular cells, and pituitary somatotrophs) (35, 36). In those cells in which Gα_s is expressed predominately, if not exclusively, from the maternal allele, it is more likely that somatic mutations of the maternal allele will have pathophysiological consequences. This is the case for sporadic growth hormone-secreting pituitary adenomas as well as patients with MAS who have growth hormone-secreting pituitary adenomas, where activating mutations of Gα_s almost always occur on the maternal allele (37, 38). By contrast, the parental origin of a gsp allele will be far less important in cells and tissues where both Gα_s alleles are expressed (e.g. bone lesions of fibrous dysplasia).

Additional transcripts are generated by GNAS using alternative first exons that are spliced to exons 2–13, but the effect of these proteins on the MAS phenotype remains uncertain. Exon 1A is located approximately 2.5 kb upstream of exon 1. Transcripts beginning with exon 1A are expressed only from the paternal allele, and are probably untranslated (39). Further upstream are two additional alternative first exons; one encodes the N-terminus of the XLα_s protein, which is expressed only from paternal alleles. XLα_s shares C-terminal sequences with Gα_s, and functions in G protein-coupled signal transduction (40). Although gsp mutations in XLα_s can affect signal transduction in vitro (41, 42), a role for gsp mutations in XLα_s in human disease has yet to be defined. The other alternative first exon is approximately 52 kb upstream of exon 1 and is expressed exclusively from the maternal allele. This exon contains the entire coding sequence for the neurosecretory protein NESP55 (43), a chromogranin-like protein that is present in secretory granules and shares no protein homology with Gα_s. Thus, activating mutations in exon 8 of GNAS would not be present in NESP55.

Third, the clinical and endocrinological features of MAS will be influenced by the particular effects of cAMP in a specific cell type. A gsp oncogene will produce the most significant consequences in those tissues in which cAMP stimulates cellular proliferation and/or hormone secretion rather than differentiation. Cyclic AMP is not mitogenic in all cell types, and in some cell types cAMP can actually inhibit growth. Moreover, even in cells in which cAMP is a strong growth stimulator, changes in the expression of other genes (44) or induction of counter-regulatory responses (such as increased cAMP phosphodiesterase activity (45–49)) could mitigate or even reverse the effects of the gsp oncogene.

Comprehensive reviews of the clinical spectrum of MAS have extended our appreciation of this unusual disorder (50–54) (Table 6.16.1). The mean age at the time of clinical diagnosis of MAS is 5.7 years, with a range of 0.7 to 11 years. Almost all patients who ultimately manifest the complete clinical triad of pigmented skin lesions, excessive endocrine function, and fibrous dysplasia will have evidence of café-au-lait skin lesions at birth. There is a 50% likelihood of precocious puberty in females by age 4 years, and a 50% likelihood of bone lesions by age 8 years.

Fibrous dysplasia of the skeleton occurs in nearly all (98%) patients with MAS, and the proportion of patients with MAS is likely to be less than 5% of all individuals with fibrous dysplasia. Although fibrous dysplasia is usually monostostic (70%), patients with MAS are more likely to have multiple fibrous dysplasia lesions (polyostotic, two-thirds of patients) than a solitary fibrous dysplasia lesion (monostotic, one-third of patients). Fibrous dysplasia typically develops during the first decade of life (Table 6.16.1), and fractures are seen to peak at age 6–10 years (55). Fibrous dysplasia seems to progress over time in most patients, with an increase in both the extent and number of bone lesions. The femur and pelvis are most commonly involved, and the shepherd’s crook deformity of the femur is a pathognomonic lesion. Spinal involvement, with progressive scoliosis, is apparently more common than originally thought (56, 57). Most affected patients will experience at least one fracture (peak age 7–12 years) and many patients will have multiple fractures. Radiographs of affected bones reveal expansile, lytic lesions with a ‘ground glass’ pattern and a scalloped cortical bone border secondary to endosteal erosion (Fig. 6.16.2). Craniofacial involvement occurs in many patients, and should be evaluated with both CT and MRI in order to demonstrate the extent of disease, and potential compressive complications of polyostotic fibrous dysplasia (PFD) (58). The marrow cavity, which usually has a cellular fatty tissue, is replaced by fibro-osseous tissue. Bone histology discloses three primary, but distinct, histological patterns, defined as Chinese writing type, sclerotic/pagetoid type, and sclerotic/hypercellular type, which are characteristically associated with the axial/appendicular skeleton, cranial bones, or gnathic bones, respectively (59).

The basis for the unusual cellular changes in fibrous dysplasia is poorly understood. Recent evidence indicates that the fibrotic areas consist of an excess of preosteogenic cells, whereas the bone formed de novo within fibrotic areas is produced by mature but abnormal osteoblasts (60). It is likely that at least some of the phenotypic changes in affected osteogenic cells result from cAMP-induced increases in protein kinase A and CREB pathways that induces overexpression of interleukin-6 and the c-fos proto-oncogene (27, 61, 62). Fos overexpression in transgenic mice results in bone lesions reminiscent of fibrous dysplasia (63). The mosaic distribution of lesions in fibrous dysplasia may also play an important pathogenic role, as close contact between transplanted normal bone cells and osteogenic cells containing the gsp mutation is necessary to reproduce the fibrous dysplasia lesion in mice (64).

Sarcomatous degeneration (e.g. osteosarcoma, fibrosarcoma, and chondrosarcoma, in descending order of frequency) occurs as a rare complication of fibrous dysplasia in MAS patients (mean age of 36 years) (65). F-18 fluorodeoxyglucose positron emission tomography may be a useful technique to identify early malignant transformation of fibrous dysplasia lesions (58, 66).

No treatment for fibrous dysplasia is entirely satisfactory. Most, but not all, studies have demonstrated that bisphosphonates can relieve bone pain, decrease bone resorption, and improve the radiological appearance (e.g. filling of lytic lesions and/or thickening of cortices) of bone lesions in about 50% of patients. Bone mineral density in affected sites is also significantly increased after treatment with pamidronate, a potent second-generation bisphosphonate which is administered intravenously (67, 68). In a series of nine patients on long-term pamidronate treatment who became resistant to this medication, a switch to intravenous zoledronic acid did not produce any substantial improvement (69).

Patients with MAS typically have one or more pigmented macules, termed café-au-lait lesions, that have irregular borders (coast of Maine) (Fig. 6.16.4). By contrast, café-au-lait skin lesions that occur in patients with neurofibromatosis (von Recklinghausen’s syndrome) have a smooth border (coast of California) (Fig. 6.16.5). The distribution of skin lesions in MAS is also characteristic (Fig. 6.16.4), consisting of an S-shaped pattern on the chest, a V-shaped pattern on the back, and a linear distribution on the extremities, which conforms to the embryological lines of ectodermal migration (i.e. lines of Blashko) and reflects the dorsoventral outgrowth of two populations of cells (20). Lesions rarely extend beyond the midline and in most patients the skin lesions tend to be on the same side of the body as the skeletal lesions. They occur most commonly on the buttocks and lumbosacral regions.

Autonomous endocrine function is common in MAS (Table 6.16.1). Precocious puberty is the most common endocrine disorder in MAS, and has been reported in over 60% of patients. Precocious puberty is a common initial manifestation of MAS in girls, and characteristically presents as thelarche and/or vaginal bleeding in a girl under 5 years of age (50). Vaginal bleeding may occur in the absence of significant breast development or pubarche. Some young girls will have seemingly regular menses and progressive pubertal development, including rapid advancement of bone age, whereas others will have irregular or intermittent bleeding that is associated with relatively normal rates of growth. The production of oestrogen appears related to the growth and involution of small ovarian cysts, and is typically not associated with follicular maturation or ovulation. Ovarian activity can undergo a spontaneous remission in some cases. Large, benign ovarian cysts may also occur (14, 15), and surgical excision may result in regression of secondary sexual characteristics until the onset of normal pubertal development. Patients typically have low or suppressed levels of serum luteinizing hormone and follicle-stimulating hormone, which fail to increase significantly after administration of gonadotropin-releasing hormone (GnRH), a characteristic of gonadotropin-independent precocious puberty (i.e. precocious ‘pseudopuberty’). Testing may be normal during intervals of apparent ovarian inactivity, however. Given the episodic nature of oestrogen production, and the poor performance characteristic of many clinical assays for oestradiol, serum concentrations of this steroid are often not elevated. Of interest, after several years of excessive sex steroid exposure some girls experience a transition to central precocious puberty, particularly those whose bone age is 11 years or greater (70–72). As adults, women with a past history of gonadotropin-independent precocious puberty may have irregular menses and reduced fertility due to continued autonomous production of oestrogen (73, 74).

Treatment of precocious puberty in girls with MAS is problematic. Therapy with GnRH analogues and superagonists is not effective unless there has been a progression to central precocious puberty (71). Treatment with aromatase inhibitors (70, 75) has been successful for short periods of time, but long-term therapy has generally been disappointing (75, 76). The efficacy of compounds with antioestrogenic activity, such as the selective oestrogen receptor modulators, tamoxifen or raloxifene, appears promising (77).

Precocious pseudopuberty also occurs in boys with MAS, but it is much less common than in young girls. Testicular biopsy reveals variable degrees of seminiferous tube development and Leydig cell hyperplasia. Testicular enlargement is generally bilateral but can be unilateral (78). Although testicular enlargement is usually associated with excessive production of testosterone and precocious puberty, occasionally the enlargement is limited to autonomous hyperfunction of Sertoli cells with no activation of Leydig cells (32). Treatment is similar to that for familial male precocious puberty due to activating mutations of the luteinizing hormone receptor (i.e. testitoxicosis) (79), and consists of the combination of an aromatase inhibitor plus an androgen receptor blocker (78, 80). In those cases where gonadotropin-independent precocious puberty leads to early activation of central puberty, the addition of a GnRH analogue may be required to arrest further pubertal development (81, 82).

Growth hormone excess is common in MAS, and may produce either gigantism or acromegaly (49, 83). The biochemical behaviour of growth hormone-producing pituitary tumours in patients with MAS appears indistinguishable from that of sporadic tumours with and without gsp mutations. Growth hormone secretion is stimulated by TRH, growth hormone releasing hormone, and sleep, and is incompletely suppressed by glucose administration. However, only 65% of MAS patients with growth hormone excess have radiographic evidence of a pituitary tumour, a much lower incidence than in sporadic cases of acromegaly (99%) (50). In addition, hyperprolactinaemia occurs in over 50% of MAS patients with elevated growth hormone levels, a frequency that is somewhat greater than occurs in patients with sporadic pituitary tumours (40%) (50).

Medical therapy with bromocriptine has been shown to reduce tumour size and hormonal secretion in many, but not all, patients (12, 44). Other medical treatments, such as long-acting octreotide and pegvisomant (84–86), appear more promising.

Hyperthyroidism and/or autonomous thyroid nodules have been identified in approximately 33% of MAS patients who underwent thyroid evaluation (50, 51, 87, 88). Radioactive iodine ablation or surgery has been used to treat thyroid nodules. The degree of hyperthyroidism is variable, and serum concentrations of TSH are typically low and thyroid stimulating immunoglobulins are undetectable. The thyroid gland will often appear normal by physical exam, but nodules are nearly always detectable by sonography.

Patients with MAS occasionally develop autonomous function of the adrenal gland and primary hypercortisolism at a young age (mean age of 4.4 years) (50). Adrenal gland histopathology reveals either nodular hyperplasia or solitary adenoma (89).

Recent analyses have documented the occurrence of additional nonendocrine features in patients with MAS that extend the clinical spectrum of the disorder. These include hypophosphataemia, hepatobiliary disease, and cardiac disease. Hypophosphataemia and/or decreased renal tubular reabsorption of phosphate occurs in over 50% of subjects with MAS, and may lead to the development of rickets or osteomalacia (90), A similar syndrome of hypophosphataemic rickets has been described in patients with fibrous dysplasia who lack other features of MAS, as well as in other patients who have various mesenchymal tumours (91), and appears due to secretion of circulating phosphaturic factors termed ‘phosphatonins’ (92, 93). FGF23 is the best characterized of the phosphatonins, and is produced by the abnormal osteogenic precursors present in fibrous dysplasia lesions. The concentration of circulating FGF23 correlates with the extent of fibrous dysplasia throughout the skeleton (94, 95). An alternative explanation for hypophosphataemia in patients with MAS is the presence of the gsp oncogene in the proximal renal tubule, where it induces increased cAMP production and an intrinsic defect in reabsorption of phosphate (96).

While neonatal jaundice in patients with MAS typical resolves, liver function enzymes typically remain mildly elevated. Liver histology varies from near normal to discrete portal fibrosis to giant cell hepatitis (97). Liver disease is due to the presence of the gsp mutation in hepatic tissue (30, 88, 97), and the degree of histological abnormality correlates with the relative amount of abnormal Gα_s protein and adenylyl cyclase activation (30). Another unusual manifestation of MAS is cardiac disease (88). Cardiac involvement in patients with MAS most commonly manifests as tachycardia and/or hypertension (88). Affected cardiac tissue contains cells with the gsp mutation (88), and it is likely that elevated levels of cAMP account directly for the abnormal cardiac function.

The diagnosis of MAS remains a clinical exercise, and is straightforward when all three cardinal features are present. However, many patients with MAS lack some features at the time of initial presentation, which makes it desirable to have a molecular test that can confirm the diagnosis. The mosaic distribution of cells bearing the GNAS mutation, and the variable number of affected cells in a tissue, makes it technically difficult to detect mutant GNAS alleles even in affected tissues, as they may represent only a small proportion of the GNAS alleles present in a DNA sample. Detection of a gsp mutant in DNA samples can be greatly enhanced by protocols that enrich the relative abundance of mutant alleles as PCR targets and thereby facilitate selective amplification. These techniques have relied upon either multiple rounds of PCR and restriction endonuclease digestion of wild type amplicons (25) or inclusion of a peptide nucleic acid (PNA) in the PCR to block amplification of wild-type GNAS targets (98, 99). The sensitivity of nested PCR and PNA-clamping appear comparable, but the nested PCR method requires more time and expense than PNA clamping (100). A recent improvement over standard PNA clamping uses a labelled PNA hybridization probe and fluorescence resonance energy transfer to allow for the direct and rapid quantification of gsp alleles with a sensitivity that allows detection in tissues that contain as few as 5% mutant cells (101). While analysis of DNA from lesional tissue affords greatest sensitivity, it is neither practical nor expedient to biopsy affected tissue(s) in all patients. Both nested PCR and PNA-clamping have been used to detect gsp mutations in peripheral blood samples (100, 102).

The detection of a gsp mutation in circulating cells from a patient with fibrous dysplasia or an isolated endocrinopathy (e.g. growth hormone-producing pituitary tumour, ovarian cysts) does not necessarily imply that the patient has MAS, however. Even with molecular demonstration of a gsp mutation, additional studies and clinical interpretation will be needed to distinguish between MAS and an isolated lesion.

On the other hand, identification of a gsp mutation can distinguish between fibrous dysplasia and similar lesion such as osteofibrous dysplasia (103), and may assist in distinguishing between atypical forms of MAS and Carney’s complex (OMIM 160980) (104–107) or Mazabraud’s syndrome (108, 109). These molecular techniques will require additional refinement and further development, however, before they can be considered as standard diagnostic tests, and at the present time no molecular technique is offered as a test for MAS in a commercial reference laboratory.

The diagnosis of MAS remains a clinical one, and requires a careful integration of physical findings, biochemical evaluation, and radiological examination. The disorder can present as a form fruste, and identification of a specific GNAS mutation in DNA from affected tissues and in many cases peripheral blood cells may one day confirm a clinical diagnosis of MAS. Finally, the genetic basis for MAS, mosaicism of a somatic gsp mutation, provides new insights into the role of imprinting as a modulator of human disease.

This work was supported in part by United States Public Health Service Grant R01 DK34281 from the NIDDK and grant RR00055 from NCRR to the Johns Hopkins General Clinical Research Center.