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The objective of this study was to describe a multiple endocrine neoplasia type 1 (MEN1) family characterized by primary hyperparathyroidism, in association with acromegaly because of ectopic growth hormone-releasing hormone (GHRH) secretion by a pancreatic neuroendocrine tumor in a young man and with a bronchial carcinoid in his mother. We investigate the clinical, radiological imaging, histopathologic findings, and therapy. An 18-year-old man successfully underwent subtotal parathyroidectomy for primary hyperparathyroidism. A subsequent genetic analysis showed a MEN1 gene mutation. Three years later, acromegaly because of ectopic GHRH secretion was diagnosed (pituitary MRI negative and elevated GHRH levels). A search for an ectopic tumor was unsuccessful and somatostatin analog therapy was started. Successively, scintigraphy with somatostatin analogs (68-Ga-DOTATOC-PET) showed three focal areas in the pancreatic tail. Distal pancreatectomy showed multiple pancreatic neuroendocrine tumors and hormonal status was normalized. Afterwards, the evaluation of the patient's mother, carrying the same mutation, indicated a primary hyperparathyroidism and a 4 cm lung mass. The patient underwent subtotal pneumonectomy and the histological analysis was consistent with the diagnosis of a typical bronchial carcinoid. In conclusion, an atypical phenotype may be recorded in MEN1 families, thus emphasizing the importance of the new imaging and surgical techniques in the diagnosis and treatment of such a rare disease.

Summary Despite intensive investigation over the past 20 years, the specific role played by individual Gi protein family members in mediating complex cellular effects is still largely unclear. Therefore, we investigated the role of specific Gi proteins in mediating somatostatin (SS) effects in somatotroph cells. Because our previous data showed that SS receptor type 5 (SST5) carrying a spontaneous R240W mutation in the third intracellular loop had a similar ability to inhibit intracellular cAMP levels to the wild-type protein but failed to mediate inhibition of growth hormone (GH) release and cell proliferation, we used this model to check specific receptor–G-protein coupling by a bioluminescent resonance energy transfer analysis. In HEK293 cells, wild-type SST5 stimulated the activation of Gαi1–3 and GαoA, B, whereas R240W SST5 maintained the ability to activate Gαi1–3 and GαoB, but failed to activate the splicing variant GαoA. To investigate the role of the selective deficit in GαoA coupling, we co-transfected human adenomatous somatotrophs with SST5 and a pertussis toxin (PTX)-resistant GαoA (GαoA(PTX-r)) protein. In PTX-treated cells, GαoA(PTX-r) rescued the ability of the selective SST5 analog BIM23206 to inhibit extracellular signal-related kinase 1/2 (ERK1/2) phosphorylation, GH secretion and intracellular cAMP levels. Moreover, we demonstrated that silencing of GαoA completely abolished SST5-mediated inhibitory effects on GH secretion and ERK1/2 phosphorylation, but not on cAMP levels. In conclusion, by analysing the coupling specificity of human SST5 to individual Gαi and Gαo subunits, we identified a crucial role for GαoA signalling in human pituitary cells.

Acrodysostosis is a rare skeletal dysplasia that is associated with multiple resistance to G protein-coupled receptor (GPCR) signaling hormones in a subset of patients. Acrodysostosis is genetically heterogeneous because it results from heterozygous mutations in PRKAR1A or PDE4D, two key actors in the GPCR-cAMP-protein kinase A pathway.Our objective was to identify the phenotypic features that distinguish the two genotypes causing acrodysostosis.Sixteen unrelated patients with acrodysostosis underwent a candidate-gene approach and were investigated for phenotypic features.All patients had heterozygous de novo mutations. Fourteen patients carried a PRKAR1A mutation (PRKAR1A patients), five each a novel PRKAR1A mutation (p.Q285R, p.G289E, p.A328V, p.R335L, or p.Q372X), nine the reported PRKAR1A p.R368X mutation; two patients harbored a mutation in PDE4D (PDE4D patients) (one novel mutation, p.A227S; one reported, p.E590A). All PRKAR1A, but none of the PDE4D mutated patients were resistant to PTH and TSH. Two PRKAR1A patients each with a novel mutation presented a specific pattern of brachydactyly. One PDE4D patient presented with acroskyphodysplasia. Additional phenotypic differences included mental retardation in PDE4D patients. In addition, we report the presence of pigmented skin lesions in PRKAR1A and PDE4D patients, a feature not yet described in the acrodysostosis entity.All PRKAR1A and PDE4D patients present similar bone dysplasia characterizing acrodysostosis. Phenotypic differences, including the presence of resistance to GPCR-cAMP signaling hormones in PRKAR1A but not PDE4D patients, indicate phenotype-genotype correlations and highlight the specific contributions of PRKAR1A and PDE4D in cAMP signaling in different tissues.

Pseudohypoparathyroidism type Ia (PHP Ia) is a rare disease characterized by an elevated parathyroid hormone due to the resistance to its action in target tissues. We report a new GNAS mutation causing PHP Ia and an atypical early-onset primary hypothyroidism. A 3-year-old boy was diagnosed with obesity, delayed pyschomotor development, and round face. The laboratory evaluation at the age of 1 year showed primary hypothyroidism, hypocalcemia, hyperphosphatemia, elevated alkaline phosphatase, and parathyroid hormone. These data led to the diagnosis of PHP Ia. Molecular analysis revealed a novel missense mutation in GNAS exon 1 (TCG→CGC, Cys3→Arg) in both the child and his mother. Although previously reported cases described delayed subclinical hypothyroidism as the more common thyroid abnormality, we report a not previously described GNAS mutation associated with an atypical early-onset primary hypothyroidism. These observations broaden the clinical spectrum of PHP Ia and its associated mutations.

A subset of over-expressed microRNAs (miRNAs) identified in parathyroid carcinomas (Ca) compared to normal glands belongs to C19MC, a cluster on chromosome 19q13.4 involved in stem cell biology and tumourigenesis. In this study, the expression of C19MC–MIR371–3 clusters and the molecular mechanisms presiding their modulation were investigated in a series of six normal parathyroids, 24 adenomas (Ad), 15 Ca and five matched metastases. The general expression levels of C19MC or MIR371–3 clusters in Ad lesions did not differ from normal glands, while they distinguished Ad from Ca at unsupervised hierarchical cluster analysis (P=0.0008). MIR517C showed the most significant difference in expression between Ca and Ad (P=0.0003) and it positively correlated with serum calcium, parathormone and tumour weight. In regard to the molecular mechanism determining C19MC cluster activation, we could detect C19MC copy number (CN) gain in ten Ca (67%) extending distal to the MIR371–3 cluster in almost all samples. Conversely, only four Ad (16%) showed C19MC amplification, with one case presenting distal genomic aberration to MIR371–3. Globally, CN variations of 19q13.4 loci were significantly associated with MIR517C up-regulation (P=0.006). Opposite to normal glands where C19MC promoter was methylated, hypomethylation occurred in 15 out of 30 analysed tumours. Though the epigenetic status did not correlate with C19MC miRNA expression levels, loss of C19MC promoter methylation was significantly associated with Ca and metastatic disease (P=0.01). In conclusion, C19MC cluster aberrations are a characteristic of Ca with respect to Ad. Altogether, these evidences point towards a role for 19q13.4 miRNA clusters as oncogenes in parathyroid tumourigenesis.

Background: Transphenoidal surgery is an effective treatment for acromegalic patients with growth hormone (GH) producing pituitary adenomas. Since acromegaly is a systemic disease which causes multiple bony alterations, we hypothesized that it could affect the sphenoid sinus anatomy. The aim of the study was to determine whether acromegalic patients have sphenoid sinus alterations with potential surgical impact. Methods: Fourty-six consecutive patients (23 acromegalics-GH group, 23 non-acromegalics-nGH group) undergoing transphenoidal surgery were included in this study. Pre-operative volumetric CT scan of the head was used to assess the following anatomic characteristics: type of sphenoid sinus (sellar, pre-sellar, conchal); number of intrasphenoid septa; number of carotid-directed septa; intercarotid distance; depth of the sphenoid sinus; depth and size of the sella. Results: The sphenoid sinus was of the pre-sellar/conchal type in 26 % of the patients with acromegaly (n = 23) versus 9 % of the patients of the nGH group (n = 23). The number of intrasphenoid septations was significantly higher in the GH group than in the nGH group (P =.03). Interestingly, the intercarotid distance was smaller in GH patients than in nGH displaying a trend toward significance (P =.05). The sphenoid bone was deeper in the GH group as compared to the nGH group (P =.01) but the distance sphenoid sinus-sella was reduced (P

The pituitary is the target of different neurohormones that have a crucial role in the control of cell differentiation, cell proliferation and hormone secretion by recognizing specific receptors belonging to the G Protein-Coupled Receptor super-family (GPCR). Evidence from in vitro studies and naturally occurring human diseases indicate that several endocrine cells, and particularly somatotrophs, recognize cAMP as a growth factor. Accordingly, mutations of the alpha subunit of the stimulatory G protein gene (GNAS) leading to the constitutive activation of adenylyl cyclase (i.e. gsp oncogene) have been recognized in a significant proportion of GH-secreting pituitary adenomas. The role of cAMP in the control of cell proliferation in selected cell types and in particular in somatotroph cells has been further confirmed by identification of genetics defect affecting the regulatory subunit IA of PKA. The role of cAMP in the control of cell proliferation as well as the crosstalk with different intracellular signalling pathways will be discussed.

Context: Dopamine agonists (DA) are the first choice treatment of prolactinomas. However, a subset of patients is resistant to DA, due to undefined dopamine D2 receptor (D2R) alterations. Recently, D2R was found to associate with filamin-A (FLNA), a widely expressed cytoskeleton protein with scaffolding properties, in melanoma and neuronal cells. Objective: The aim of the study was to investigate the role of FLNA in D2R expression and signaling in human tumorous lactotrophs and rat MMQ and GH3 cells. Design: We analyzed FLNA expression in a series of prolactinomas by immunohistochemistry and Western blotting.Weperformed FLNA silencing or transfection experiments in cultured cells from DA-sensitive or -resistant prolactinomas and in MMQ and GH3 cells, followed by analysis of D2R expression and signaling. Results: We demonstrated reduced FLNA and D2R expression in DA-resistant tumors. The crucial role of FLNA on D2R was demonstrated by experiments showing that: 1) FLNA silencing in DA-sensitive prolactinomas resulted in 60% reduction of D2R expression and abrogation of DA-induced inhibition of prolactin release and antiproliferative signals, these results being replicated in MMQ cells that endogenously express FLNA and D2R; and 2) FLNA overexpression in DA-resistant prolactinomas restored D2R expression and prolactin responsiveness to DA, whereas this manipulation was ineffective in GH3 cells that express FLNA but not D2R. No alteration in FLNA promoter methylation was detected, ruling out the occurrence of epigenetic FLNA silencing in DA-resistant prolactinomas. Conclusions: These data indicate that FLNA is crucial for D2R expression and signaling in lactotrophs, suggesting that the impaired response to DA may be related to the reduction of FLNA expression in DA-resistant prolactinomas. Copyright © 2012 by The Endocrine Society.

Context: The influence of full-length GH receptor (GHR) and exon 3-deleted GHR (d3GHR) on responsiveness to pegvisomant (PEG-V) in acromegalic patients is uncertain. Objective: The aim of the study was to assess the distribution of GHR genotypes in a large series of patients on PEG-V therapy and their influence on treatment efficacy and adverse effects. Design and Setting: A cross-sectional multicenter pharmacogenetic study was conducted in 16 Italian endocrinology centers of major universities and tertiary care hospitals. Patients: The study included 127 acromegalic patients enrolled from 2009 to 2010 not cured by previous surgery, radiotherapy, and long-acting somatostatin (SST) analogs, treated with PEG-V. Intervention and Main Outcome Measure: Sixty-three of 127 patients received combined PEG-V + SST analog therapy. Clinical and hormonal data at diagnosis and before and during PEG-V therapy were inserted in a database. GHR exon 3 deletion and other polymorphisms were genotyped by the coordinator center. Differences in PEG-V dosage required for IGF-I normalization and occurrence of adverse effects between carriers and noncarriers of GHR variants were evaluated. Results: d3GHR variants were not in Hardy-Weinberg equilibrium (P = 0.008). No association of these variants with PEG-V dose required for IGF-I normalization, adverse effects occurrence, and tumor regrowth was found in patients on PEG-V and on PEG-V + SST analog treatment. Similar data were obtained considering the GHR variant rs6180. Conclusions: This study did not confirm a better response of d3GHR to PEG-V treatment in acromegaly. Other studies are needed to determine whether deviation from Hardy-Weinberg equilibrium may indicate an association of d3GHR genotype with poor response to usual treatments.

The term pseudohypoparathyroidism (PHP) indicates a group of heterogeneous disorders whose common feature is represented by impaired signaling of various hormones (primarily PTH) that activate cAMP-dependent pathways via Gsα protein. The two main subtypes of PHP, PHP type Ia, and Ib (PHP-Ia, PHP-Ib) are caused by molecular alterations within or upstream of the imprinted GNAS gene, which encodes Gsα and other translated and untranslated products.A PubMed search was used to identify the available studies (main query terms: pseudohypoparathyroidism; Albright hereditary osteodystrophy; GNAS; GNAS1; progressive osseous heteroplasia). The most relevant studies until February 2011 have been included in the review.Despite the first description of this disorder dates back to 1942, recent findings indicating complex epigenetic alterations beside classical mutations at the GNAS complex gene, pointed out the limitation of the actual classification of the disease, resulting in incorrect genetic counselling and diagnostic procedures, as well as the gap in our actual knowledge of the pathogenesis of these disorders. This review will focus on PHP type I, in particular its diagnosis, classification, treatment, and underlying molecular alterations.

Context The cutoff value of nadir GH after an oral glucose tolerance test (OGTT) used to define disease remission in acromegaly is higher than that observed in healthy subjects. However, it is uncertain whether the impaired GH inhibition might be related to subtle abnormalities of GH secretion or to functional and/or anatomical hypothalamic-pituitary disconnection due to tumor per se or treatments. Objective The objective of the study was to evaluate the impact of pituitary disorders other than acromegaly on GH response to OGTT. Design, Subjects, and Methods Thirty-three patients (24 females and nine males, aged 50.1 ± 12.3 yr, 13 operated and two irradiated) with various hypothalamic-pituitary disorders (HPDs), 45 healthy subjects (controls), and 42 cured acromegalic patients matched for sex, age. and body mass index were investigated. All subjects were studied for IGF-I levels and GH levels before and during the OGTT. Results In HPD patients mean postglucose nadir GH levels were 0.11 ± 0.08 μg/liter without any difference between patients treated with neurosurgery and/or radiotherapy and untreated and between patients with and without pituitary stalk alterations and/or hyperprolactinemia. Mean nadir GH values were similar in HPD patients and controls (0.11 ± 0.08 vs. 0.08 ± 0.08 μg/liter, P = 0.23) and lower than those found in cured acromegalic patients (0.18 ± 0.13 μg/liter, P = 0.02), although there was an overlapping in about half of patients. Conclusions Hypothalamic control of glucose-mediated GH suppression is not perturbed in patients with HPD. These data indicate that defective GH suppression to glucose that is found in acromegaly is unlikely to reflect a lack of integrity of hypothalamic function.

Patients with McCune-Albright syndrome (MAS), characterized primarily by hyperpigmented skin lesions, precocious puberty, and fibrous dyslasia of bone, carry postzygotic heterozygous mutations of GNAS causing constitutive cAMP signaling. GNAS encodes the α-subunit of the stimulatory G protein (Gsα), as well as a large variant (XLαs) derived from the paternal allele. The mutations causing MAS affect both GNAS products, but whether XLαs, like Gsα, can be involved in the pathogenesis remains unknown. Here, we investigated biopsy samples from four previously reported and eight new patients with MAS. Activating mutations of GNAS (Arg201 with respect to the amino acid sequence of Gsα) were present in all the previously reported and five of the new cases. The mutation was detected within the paternally expressed XLαs transcript in five and the maternally expressed NESP55 transcript in four cases. Tissues carrying paternal mutations appeared to have higher XLαs mRNA levels than maternal mutations. The human XLαs mutant analogous to Gsα-R201H (XLαs-R543H) showed markedly higher basal cAMP accumulation than wild-type XLαs in transfected cells. Wild-type XLαs demonstrated higher basal and isoproterenol-induced cAMP signaling than Gsα and co-purified with Gβ1γ2 in transduced cells. XLαs mRNA was measurable in mouse calvarial cells, with its level being significantly higher in undifferentiated cells than those expressing preosteoblastic markers osterix and alkaline phosphatase. XLαs mRNA was also expressed in murine bone marrow stromal cells and preosteoblastic MC3T3-E1 cells. Our findings are consistent with the possibility that constitutive XLαs activity adds to the molecular pathogenesis of MAS and fibrous dysplasia of bone.

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) and resistance toward PTH and additional hormones, whereas PHP-Ib patients do not have AHO, and hormone resistance appears to be limited to PTH and TSH. Recently, methylation defects have been detected in few patients with PHP and mild AHO, indicating a molecular overlap between the two forms.The aim of the study was to screen patients with clinically diagnosed PHP-Ia for methylation defects and to investigate the presence of correlations between the molecular findings and AHO severity.We investigated differential methylation of GNAS regions and STX16 microdeletions in genomic DNA from 40 patients with sporadic AHO and multihormone resistance, with no mutations in Gsalpha-coding GNAS exons.Molecular analysis showed GNAS cluster imprinting defects in 24 of the 40 patients analyzed. No STX16 deletion was detected. The presence of imprinting defects was not associated with the severity of AHO or with specific AHO signs.We report the largest series of the literature of patients with clinical AHO and multihormone resistance and no mutation in the Gsalpha gene. Our findings of frequent GNAS imprinting defects further confirm the existence of an overlap between molecular and clinical features of PHP-Ia and PHP-Ib and highlight the necessity of a new clinical classification of the disease that takes into account the recent knowledge on the molecular basis underlying these defects.

Since the identification of GH deficiency due to resistance to GHRH in patients with pseudohypoparathyroidism type Ia (PHP-Ia), no study investigated the effects of recombinant human GH (rhGH) therapy on height velocity (HV) in these patients.To address this question, eight prepubertal PHP-Ia children with GH deficiency (seven girls and one boy, aged 5.8-12 yr) underwent a 3- to 8-yr treatment with rhGH. Height and HV were measured before and at 6-month intervals during therapy. Nine sex- and age-matched children with idiopathic GH deficiency were monitored during rhGH therapy for comparison.In PHP-Ia children, height sd scores increased from -2.4 ± 0.58 to -1.8 ± 0.47 (P = 0.04) after 12 months, this increase being maintained after the second (-1.6 ± 0.6) and third (-1.15 ± 0.6) year of therapy, similarly to what recorded in children with idiopathic GH deficiency. The HV and HV sd scores after 3 yr maintained a significant increase from 3.5 ± 0.6 to 7.0 ± 0.9 cm/yr (P0.0001) and from -2.8 ± 0.8 to +2.2 ± 1.0 (P0.0001), respectively. Six patients treated for 4-8 yr had a reduced pubertal spurt and did not improve their near-adult height, with the only exception of one patient in whom estrogen production was blocked by GnRH analogs.We report the first study on the efficacy of rhGH replacement therapy in prepubertal children with PHP-Ia and provide indication that treatment of GH deficiency should be started soon due to the rather limited time window for a potentially effective therapy.

Cardiac myxomas are rare tumors that usually occur as sporadic lesions or, more rarely, in the familial form, mostly in the context of Carney complex (CNC). The molecular basis for the development of cardiac myxomas is unclear. However, somatic activating mutations in the GNAS1 gene (the gsp oncogene) are detected in the myocardium of McCune-Albright syndrome patients while germ-line mutations in the PRKAR1A gene are associated with CNC and familial myxomas. We investigated the presence of activating missense mutations in the GNAS1 gene as well as of inactivating mutations in PRKAR1A in 29 sporadically occurring cardiac myxomas. No gsp and no PRKAR1A mutations were found by direct sequencing of PCR products amplified from tumoral DNA. This is the first study including a large series of sporadic, isolated cardiac myxomas and showing that these cardiac neoplasms do not share the same mutations found in familial forms.

OBJECTIVE—In human adipocytes, the cAMP-dependent pathway mediates signals originating from β-adrenergic activation, thus playing a key role in the regulation of important metabolic processes, i.e., lipolysis and thermogenesis. Cyclic AMP effects are mainly mediated by protein kinase A (PKA), whose R2B regulatory isoform is the most expressed in mouse adipose tissue, where it protects against diet-induced obesity and fatty liver development. The aim of the study was to investigate possible differences in R2B expression, PKA activity, and lipolysis in adipose tissues from obese and nonobese subjects. RESEARCH DESIGN AND METHODS—The expression of the different PKA regulatory subunits was evaluated by immunohistochemistry, Western blot, and real-time PCR in subcutaneous and visceral adipose tissue samples from 20 nonobese and 67 obese patients. PKA activity and glycerol release were evaluated in total protein extract and adipocytes isolated from fresh tissue samples, respectively. RESULTS—Expression techniques showed that R2B was the most abundant regulatory protein, both at mRNA and protein level. Interestingly, R2B mRNA levels were significantly lower in both subcutaneous and visceral adipose tissues from obese than nonobese patients and negatively correlated with BMI, waist circumference, insulin levels, and homeostasis model assessment of insulin resistance. Moreover, both basal and stimulated PKA activity and glycerol release were significantly lower in visceral adipose tissue from obese patients then nonobese subjects. CONCLUSIONS—Our results first indicate that, in human adipose tissue, there are important BMI-related differences in R2B expression and PKA activation, which might be included among the multiple determinants involved in the different lipolytic response to β-adrenergic activation in obesity.

The genesis of pituitary tumors is still under debate. Although these neoplasia are monoclonal in origin, mutations of GNAS1, the gene encoding the α subunit of Gs is the only mutational change unequivocally associated with GH-secreting adenomas. In addition, multiple events, including the overexpression of cell cycle regulators, growth factors, and stimulatory hormones together with epigenetic disruption of genes with antioncogenic properties, frequently occur in pituitary tumors; their relative importance is still uncertain.

Dopamine-agonist cabergoline (CB) reduces prolactin (PRL) secretion and tumor size in 80% of patients with prolactin-secreting adenomas (PRL-omas) by binding type 2 dopamine receptor (DRD2). The mechanisms responsible for resistance to CB remain largely unknown. To assess the association of DRD2 with sensitivity to CB, TaqI-A1/A2, TaqI-B1/B2, HphI-G/T and NcoI-C/T genotypes were determined in a cross-sectional retrospective study, including 203 patients with PRL-oma. DRD2 alleles frequencies did not differ between patients and 212 healthy subjects. Conversely, NcoI-T allele frequency was higher in resistant rather than responsive patients, considering both PRL normalization (56.6 vs 45.3%, P=0.038) and tumor shrinkage (70.4 vs 41.4%, P=0.006). Finally, [TaqI A1-/TaqI B1-/HphI T-/NcoI T-] haplotype was found in 34.5% of patients normalizing PRL with < or =3 mg/week of CB vs 11.3% of resistants (P=0.021). In conclusion, resistance to CB was associated with DRD2 NcoI-T+ allele, consistent with evidence suggesting that this variant may lead to reduction and instability of DRD2 mRNA or protein.

Background Macroprolactinaemia may represent a relevant cause of misdiagnosis, unnecessary investigation and inappropriate treatment. The aim of this study was to investigate the clinical and neuroradiological characteristics of patients with and without macroprolactinaemia and to evaluate the impact of macroprolactin determination on the diagnostic work-up of hyperprolactinaemic patients. Materials and methods Retrospective analysis in 135 consecutive hyperprolactinaemic patients (111 women and 24 men; mean age 37 ± 11·6 years) whose archived sera were subsequently tested for macroprolactin. Recoveries ≤ 40% after polyethylene glycol precipitation were indicative of macroprolactinaemia. Results Macroprolactin, entirely explaining biochemical hyperprolactinaemia, was found in 42·2% of patients, a third of whom presented with signs and symptoms of hyperprolactinaemia. Determination of macroprolactin changed the initial diagnosis in a consistent proportion of patients. In particular, idiopathic hyperprolactinaemia, initially diagnosed in 41 patients, was then excluded in 28 of them. Diagnosis of prolactin-secreting pituitary microadenoma shifted to non-secreting pituitary microadenoma in 10 of 49 patients, while in all patients with prolactin-secreting pituitary macroadenoma or hyperprolactinaemia due to stalk deafferentation the presence of macroprolactin was excluded and the initial diagnosis confirmed. Finally, macroprolactin was present in the majority of patients with magnetic resonance imaging (MRI) scans suggestive for primary empty sella (4 of 5 women) or pituitary hyperplasia (12 of 17 women, 3 of 3 men). Collectively, about half of subjects with macroprolactinaemia showed variable MRI abnormalities. Conclusions The presence of macroprolactin was a relevant cause of misdiagnosis in patients with hyperprolactinaemia. However, due to the unexpected high frequency of pituitary abnormalities observed in the present series, we suggest that the diagnostic algorithm of hyperprolactinaemic states should include both polyethylene glycol precipitation test and MRI imaging.

Somatostatin exerts antisecretive and antiproliferative effects on different endocrine cells by acting through a family of G protein-coupled receptors that includes five subtypes (SST1-5). Normal human pituitary and pituitary adenomas have been shown to express almost all SST subtypes, with the exception of SST4. Consistent with the observation that octreotide and other somatostatin analogs bind to SST2 and SST5 with high affinity, these genes have been screened for quantitative/qualitative abnormalities in tumors removed from patients with poor responsiveness to somatostatin analogs treatment. Data obtained in GH-secreting adenomas suggested that resistance to octreotide was frequently associated with low expression of SST2 mRNA, although other authors failed to confirm this finding. To date, the only mutational change involving SST2 and SST5 is the Arg to Trp substitution in codon 240 of the SST5 gene that was found in one acromegalic patient resistant to octreotide. Similarly, loss of heterozygosis at SST5 gene locus in pituitary adenomas has been described in individual tumors. In recent years, molecular studies investigated the possible association of gene polymorphisms and susceptibility to diseases and/or resistance to drugs. As far as polymorphic variants of SST genes are concerned, a possible role of SST5 C1004T and T-461C alleles in influencing GH and IGF-I levels in patients with acromegaly has been proposed. Nevertheless, polymorphic variants in SST2 and SST5 genes seem to have a minor, if any, role in determining the different responsiveness to somatostatin analogs in patients with acromegaly.

The four regulatory subunits (R1A, R1B, R2A, R2B) of protein kinase A (PKA) are differentially expressed in several cancer cell lines and exert distinct roles in growth control. Mutations of the R1A gene have been found in patients with Carney complex and in a minority of sporadic primary pigmented nodular adrenocortical disease (PPNAD). The aim of this study was to evaluate the expression of PKA regulatory subunits in non-PPNAD adrenocortical tumors causing ACTH-independent Cushing's syndrome and to test the impact of differential expression of these subunits on cell growth. Immunohistochemistry demonstrated a defective expression of R2B in all cortisol-secreting adenomas ( n = 16) compared with the normal counterpart, while both R1A and R2A were expressed at high levels in the same tissues. Conversely, carcinomas ( n = 5) showed high levels of all subunits. Sequencing of R1A and R2B genes revealed a wild type sequence in all tissues. The effect of R1/R2 ratio on proliferation was assessed in mouse adrenocortical Y-1 cells. The R2-selective cAMP analogue 8-Cl-cAMP dose-dependently inhibited Y-1 cell proliferation and induced apoptosis, while the R1-selective cAMP analogue 8-HA-cAMP stimulated cell proliferation. Finally, R2B gene silencing induced up-regulation of R1A protein, associated with an increase in cell proliferation. In conclusion, we propose that a high R1/R2 ratio favors the proliferation of well differentiated and hormone producing adrenocortical cells, while unbalanced expression of these subunits is not required for malignant transformation.

In recent years the demonstration that human pituitary adenomas are monoclonal in origin provides further evidence that pituitary neoplasia arise from the replication of a single mutated cell in which growth advantage results from either activation of proto-oncogenes or inactivation of tumor suppressor genes. Mutations in common oncogenes and tumor suppressor genes are only exceptionally involved in pituitary tumors. Since pituicytes may proliferate in response to hypothalamic neurohormones, locally produced growth factors and peripheral hormones, it has been speculated that dysregulation of the signaling molecules that constitute these pathways may confer growth advantage to the target cell, finally resulting in tumor formation. The only mutational change so far recognized to be unequivocally associated with pituitary tumors occur in the Gsα gene (GNAS1) and cause constitutive activation of the cAMP-dependent pathway. However, other components of pituitary-specific pathways are frequently altered in their expression and activity. This review will focus on the possible impact of G proteins and other components of hormone signaling on pituitary tumorigenesis.

The study investigated cyclin D1 regulation by growth factors and calcium sensing receptor (CaSR) in human tumoral parathyroid cells. Basic fibroblast and epidermal growth factors increased cyclin D1 and phosphorylated extracellular signal-regulated kinases (pERK1/2) levels that were both efficiently inhibited by CaSR agonists. By contrast, in growth factors-free medium cyclin D1 levels were either unaffected or stimulated by CaSR activation independently from ERK1/2 pathway. Transforming growth factor beta (TGFbeta) reduced cyclin D1 levels in the majority of tumors, this effect being not influenced by CaSR activation and menin expression levels. In conclusion, in parathyroid tumors cyclin D1 expression was modulated by growth factors and CaSR activation. These data further support the oncogenic role of cyclin D1, which resulted to be target for stimulation by bFGF and EGF and inhibition by CaSR and TGFbeta signalling in the parathyroid.

Heterozygous inactivating mutations in the Gs alpha gene cause Albright's hereditary osteo-dystrophy (AHO). Consistent with the observation that only maternally inherited mutations lead to resistance to hormone action (pseudohypoparathyroidism type Ia [PHP-Ia), recent studies have provided evidence for a predominant maternal origin of Gs alpha transcripts in endocrine organs, such as thyroid, gonad and pituitary. Accordingly, patients with PHP-Ia display variable degrees of resistance to parathyroid hormone (PTH), thyroid stimulating hormone (TSH), gonadotropins and growth hormone (GH) releasing hormone (GHRH). Although the incidence and the clinical and biochemical characteristics of PTH and TSH resistance have been widely investigated and described, the cause and significance of the reproductive dysfunction in AHO is still poorly understood. The clinical finding of alterations of GH secretion in these patients was described for the first time only 2 years ago. The present report briefly reviews the literature focusing on the actual knowledge about these last two subjects.

Objective: Mutations in the gene coding for the orphan nuclear receptor DAX1 cause X-linked adrenal hypoplasia congenita (AHC). Affected boys usually present with primary adrenal failure in early infancy or childhood. Impaired sexual development due to hypogonadotropic hypogonadism becomes manifest at the time of puberty. Moreover, evidence from Dax1 knockout mice and a limited number of patients with AHC, suggests that mutations in DAX1 may directly cause abnormalities in spermatogenesis. The aim of this study was to characterize clinically and genetically five patients with AHC. Design: DNA sequencing analysis, endocrine testing, testicular ultrasound and semen analysis with 1-year follow-up after gonadotropin treatment. Methods: We report on five men with classic AHC manifestations. Genomic DNA was extracted from patients’ peripheral blood leukocytes and the coding region, splice sites, and promoter (−240 bp) region of DAX1 were directly sequenced. Results: Three known and two novel mutations were detected in the DAX1 coding sequence in these patients. Semen analysis was performed in four of the five patients and showed azoospermia. Twelvemonth treatment with gonadotropins did not restore fertility in these patients. All patients showed a normal testicular Doppler ultrasound, in contrast with that observed in Dax1-deficient mice, which display abnormalities in the rete testis. Conclusions: These cases further expand the number of DAX1 mutations reported in the literature, as well as our clinical knowledge of this rare disease.

In recent years the demonstration that human pituitary adenomas are monoclonal in origin provides further evidence that pituitary neoplasia arise from the replication of a single mutated cell in which growth advantage results from either activation of proto-oncogenes or inactivation of tumor suppressor genes. However, with the exception of one RAS mutation identified in a single unusually aggressive prolactinoma resistant to dopaminergic inhibition that resulted to be lethal, no mutational changes have been so far detected in prolactinomas. In the absence of genetic changes, modifications in the level of expression of oncogenes or tumor suppressor genes have been detected in these tumors, although it is unknown whether these changes have a causative role or are a secondary event. Indeed, our knowledge on the molecular events involved in lactotroph proliferation is even more limited in comparison to the other tumor types, since these tumors are very infrequently surgically removed and therefore available for molecular biology studies. In this respect, it is worth noting that the molecular and biological abnormalities so far described in prolactinomas mainly concern aggressive and atypical tumors and likely do not apply to the typical prolactinomas, that are characterized by good response to medical treatment and a very low growth rate.

Heterozygous inactivating mutations in the Gs alpha gene cause Albright's hereditary osteo-dystrophy (AHO). Consistent with the observation that only maternally inherited mutations lead to resistance to hormone action (pseudohypoparathyroidism type Ia [PHP-Ia), recent studies have provided evidence for a predominant maternal origin of Gs alpha transcripts in endocrine organs, such as thyroid, gonad and pituitary. Accordingly, patients with PHP-Ia display variable degrees of resistance to parathyroid hormone (PTH), thyroid stimulating hormone (TSH), gonadotropins and growth hormone (GH) releasing hormone (GHRH). Although the incidence and the clinical and biochemical characteristics of PTH and TSH resistance have been widely investigated and described, the cause and significance of the reproductive dysfunction in AHO is still poorly understood. The clinical finding of alterations of GH secretion in these patients was described for the first time only 2 years ago. The present report briefly reviews the literature focusing on the actual knowledge about these last two subjects.

Previous studies indicate that nuclear factor kappaB (NF-κB) transcription factor is deregulated and overexpressed in several human neoplasias. The aim of this study was to test the hypothesis that the NF-κB pathway may be involved in parathyroid tumorigenesis. For this purpose, we determined the level of NF-κB activity, evaluated as phosphorylation of the transcription subunit p65, its modulation by specific and non-specific agents and its impact on cyclin D1 expression. Phosphorylated p65 levels present in parathyroid neoplasias (n = 13) were significantly lower than those found in normal tissues (n = 3; mean optical density (OD) 0.19 ± 0.1 vs 0.4 ± 0.1, P = 0.007), but there was no significant difference between adenomas and secondary and multiple endocrine neoplasia type 1 (MEN1)-related hyperplasia. Conversely, MEN2A (Cys634Arg)-related parathyroid samples showed extremely high levels of phosphorylated p65 that exhibited a nuclear localization at immunohistochemistry (n = 3). Phosphorylated p65 levels negatively correlated with menin expression (r2 = 0.42, P = 0.05). Tumor necrosis factor-α (TNFα) caused a significant increase in phosphorylated p65 levels (183 ± 13.8% of basal) while calcium sensing receptor (CaR) agonists exerted a significant inhibition (19.2 ± 3.3% of basal). Although TNFα was poorly effective in increasing cyclin D1 expression, NF-κB blockade by the specific inhibitor BAY11-7082 reduced FCS-stimulated cyclin D1 by about 60%. Finally, the inhibitory effects of CaR and BAY11-7082 on cyclin D1 expression were not additive – by blocking NF-κB CaR activation did not induce a further reduction in cyclin D1 levels. In conclusion, the study demonstrated that in parathyroid tumors: (1) p65 phosphorylation was dramatically increased by RET constitutive activation and was negatively correlated with menin expression, (2) p65 phosphorylation was increased and reduced by TNFα and CaR agonists respectively, and (3) blockade of the NF-κB pathway caused a significant decrease in cyclin D1 expression.

Alterations in cAMP signaling have been identified as a cause of endocrine neoplasia. In particular, activating mutations of the G(s)alpha gene and protein kinase A (PKA) overactivity due to low expression of PKA regulatory subunit 1A (R1A) have been implicated in somatotroph proliferation.The objective of this study was to evaluate the effects of cAMP-PKA cascade activation in nonfunctioning pituitary adenomas (NFPA).By immunohistochemistry, R1A, R2A, and R2B expression was evaluated in cells obtained from eight surgically removed NFPA positive for gonadotropins. Cyclin D1 expression and ERK1/2 activity were analyzed under basal conditions and after cAMP-PKA cascade activation.Immunohistochemistry studies demonstrated a low R1/R2 ratio in all NFPA. Additional unbalance of R1/R2 ratio by 8-chloroadenosine cAMP (8-Cl-cAMP) and direct adenylyl cyclase stimulation by forskolin did not increase cyclin D1 expression or ERK1/2 activity in five NFPA (group 1), but even caused 74 +/- 15% and 85 +/- 13% inhibitions of cyclin D1 and ERK1/2 activity, respectively, in the remaining NFPA (group 2). Moreover, in group 2, PKA blockade by the specific inhibitor PKI increased cyclin D1 expression (96 +/- 25% over basal) and ERK1/2 activity (116 +/- 28% over basal).These data show that in contrast with what was previously observed in transformed somatotrophs, activation of the cAMP-PKA pathway did not generate proliferative signals in tumoral cells of the gonadotroph lineage, and in a subset of tumors even exerted a tonic inhibitory effect, thus confirming a different role for the cAMP-mediated pathway in promoting proliferation in the pituitary.

Seladin-1 (from selective Alzheimer's disease indicator-1) is a recently discovered gene that has been found to be down-regulated in brain regions affected by Alzheimer's disease. Seladin-1 effectively protects neurons against beta-amyloid-mediated toxicity and prevents apoptosis via inhibition of the activation of caspase-3, a key mediator of the apoptotic cascade. Although seladin-1 is expressed in the pituitary gland, no study addressed the expression or the function of this gene in pituitary adenomas.The aim of the present study was to determine the expression level of the seladin-1 gene in pituitary tumors, i.e. GH-secreting and nonfunctioning pituitary adenomas (NFPA), and to determine whether differential expression might be associated with different somatostatin (sst)-induced apoptosis.We found by quantitative real-time RT-PCR that the expression level of seladin-1 was significantly higher in NFPA (n = 21) than in GH-secreting adenomas (n = 30; mean +/- se, 25.69 +/- 6.39 vs. 8.02 +/- 2.68 pg/microg total RNA; P = 0.006). Although the amount of activated caspase-3 did not differ between the two groups of tumors, in primary cell cultures, octreotide was able to increase apoptosis, evaluated by the level of cleaved cytokeratin 18 and the presence of apoptotic nuclei, in GH-secreting adenomas, but not in NFPA. This different response was not attributable to differences in the amount of transcript of sst receptors 2 and 5, which was similar in the two groups of tumors.Our results suggest that differential seladin-1 expression in pituitary adenomas may be associated with a different apoptotic response to sst analogs.

Heterozygous loss of function mutations in human PKAR1A gene (PRKAR1A) have been identified in patients with Carney complex (CNC), an autosomal dominant familial multiple neoplasia syndrome displaying different endocrine tumors, including adrenocortical tumors, GH-secreting pituitary tumors and thyroid adenomas. Although PRKAR1A is encoded by a single gene, it is transcribed from at least two different promoters, adjacent to different first non-coding exons (1a and 1b), giving rise to alternately spliced transcripts coding for identical proteins. The separate regulation of the two distinct promoters and the presence of multiple alternatively spliced first exons suggest a complex mechanism of PRKAR1A expression regulation. In order to investigate the relative expression of the two mRNA transcripts (1a and 1b) in human adult endocrine tissues involved in the determination of CNC phenotype, we selected 17 pituitary, 20 adrenal and seven thyroid tissues from tumoral and peri-tumoral lesion samples. Expression of the two transcripts was evaluated by semi-quantitative RT-PCR and real-time RT-PCR. This study first reports that human pituitary and thyroid tissues show a similar expression of the two transcripts, whereas in adrenal tissues transcript 1b is the most abundant one.

Adiponectin (ApN), an adipocytokine expressed in adipocytes with antidiabetic and antiatherogenic actions, has been detected in cord blood, suggesting a putative role in intrauterine fetal development. The aim of this study was to confirm the presence of ApN in the fetal circulation and directly investigate ApN expression in fetal tissues. The study showed high ApN levels in umbilical venous blood from fetuses [n = 44; 31.2 ± 14.1 (sd) mg/liter in umbilical vs. 8.4 ± 4.0 in maternal circulation (P < 0.0001)] that positively correlated with gestational age. By using RT-PCR, Western blotting, and immunohistochemistry, ApN was detected in several fetal tissues at mid- and late gestation (from 14 to 36 wk) but not in the placenta. ApN was expressed in tissues of mesodermic origin, i.e. brown and white adipocytes, skeletal muscle fibers of diaphragm and iliopsoas, smooth muscle cells of small intestine and arterial walls, perineurium and renal capsule, and tissues of ectodermal origin, i.e. epidermis and ocular lens. The distribution of ApN expression in nonadipose tissues showed a general decline during the progression of gestation. The unexpected pattern of ApN expression in the human fetus may account for the high ApN levels in cord blood and predicts novel roles for ApN during fetal development.

The two regulatory subunits (R1 and R2) of protein kinase A (PKA) are differentially expressed in cancer cell lines and exert diverse roles in growth control. Recently, mutations of the PKA regulatory subunit 1A gene (PRKAR1A) have been identified in patients with Carney complex. The aim of this study was to evaluate the expression of the PKA regulatory subunits R1A, R2A, and R2B in a series of 30 pituitary adenomas and the effects of subunit activation on cell proliferation. In these tumors, neither mutation of PRKAR1A nor loss of heterozygosity was identified. By real-time PCR, mRNA of the three subunits was detected in all of the tumors, R1A being the most represented in the majority of samples. By contrast, immunohistochemistry documented low or absent R1A levels in all tumors, whereas R2A and R2B were highly expressed, thus resulting in an unbalanced R1/R2 ratio. The low levels of R1A were, at least in part, due to proteasome-mediated degradation. The effect of the R1/R2 ratio on proliferation was assessed in GH3 cells, which showed a similar unbalanced pattern of R subunits expression, and in growth hormone-secreting adenomas. The R2-selective cAMP analog 8-Cl cAMP and R1A RNA silencing, stimulated cell proliferation and increased Cyclin D1 expression, respectively, in human and rat adenomatous somatotrophs. These data show that a low R1/R2 ratio promoted proliferation of transformed somatotrophs and are consistent with the Carney complex model in which R1A inactivating mutations further unbalance this ratio in favor of R2 subunits. These results suggest that low expression of R1A protein may favor cAMP-dependent proliferation of transformed somatotrophs.

Mutations of the Gs gene inherited from the mother lead to pseudohypoparathyroidism (PHP) type Ia (PHP Ia), in which Albright’s hereditary osteodistrophy is associated to resistance to the action of different hormones, whereas the same mutations inherited from the father lead to isolated Albright’s hereditary osteodistrophy [pseudo-PHP (PPHP)]. Accordingly, it has been suggested that Gs is under tissue-specific imprinting control, and recent studies provided evidence for a predominant maternal origin of Gs transcripts in different endocrine organs involved in the PHP Ia phenotype. To establish whether Gs is imprinted also in tissues that are site of alteration both in PHP Ia and PPHP, we selected 20 bone and 10 adipose tissue samples, which were heterozygous for a known polymorphism in exon 5. Expression from both parental alleles was evaluated by RT-PCR and enzymatic digestion of the resulting fragments. By this approach, the great majority of the samples analyzed showed an equal expression of the two alleles. Our results provide evidence for the absence of Gs imprinting in human bone and fat and suggest that the clinical finding of osteodystrophy and obesity in PHP Ia and PPHP patients despite the presence of a normal Gs allele is likely due to Gs haploinsufficiency in these tissues. (J Clin Endocrinol Metab 89: 6316–6319, 2004)

Mutations in the orphan nuclear receptor DAX1 (NR0B1) cause X-linked adrenal hypoplasia congenital (AHC), a disorder characterized by primary adrenal failure, hypogonadotropic hypogonadism. and azoospermia. We tested the hypothesis that DAX1 somatic mutations in human testis may cause azoospermia. DAX1 sequencing analysis in 15 testicular biopsy samples from men with idiopathic nonobstructive azoospermia did not reveal mutations in the coding region of the gene. We conclude that somatic abnormalities in DAX1 are absent or uncommon in these patients.

PTX3 is a prototypic long pentraxin that plays a non-redundant role in innate immunity against selected pathogens and in female fertility. Here, we report that the infertility of Ptx3–/– mice is associated with severe abnormalities of the cumulus oophorus and failure of in vivo, but not in vitro, oocyte fertilization. PTX3 is produced by mouse cumulus cells during cumulus expansion and localizes in the matrix. PTX3 is expressed in the human cumulus oophorus as well. Cumuli from Ptx3–/– mice synthesize normal amounts of hyaluronan (HA), but are unable to organize it in a stable matrix. Exogenous PTX3 restores a normal cumulus phenotype. Incorporation in the matrix of inter-α-trypsin inhibitor is normal in Ptx3–/– cumuli. PTX3 does not interact directly with HA, but it binds the cumulus matrix hyaladherin tumor necrosis factor α-induced protein 6 (TNFAIP6, also known as TSG6) and thereby may form multimolecular complexes that can cross-link HA chains. Thus, PTX3 is a structural constituent of the cumulus oophorus extracellular matrix essential for female fertility.

Activating mutations of the Gsalpha gene are detected in different endocrine tumors, such as GH-secreting adenomas and toxic thyroid adenomas, and in hyperfunctioning glands from patients with McCune-Albright syndrome (MAS). There is increasing evidence that the Gsalpha gene is subjected to imprinting control and that Gsalpha imprinting plays a key role in the pathogenesis of different human diseases. The aim of this study was to investigate the presence of a parent specificity of Gsalpha mutations in 10 patients affected with MAS and 12 isolated tumors (10 GH-secreting adenomas, one toxic thyroid adenoma, and one hyperfunctioning adrenal adenoma). The parental origin of Gsalpha mutations was assessed by evaluating NESP55 and exon 1A transcripts, which are monoallelically expressed from the maternal and paternal alleles, respectively. By this approach, we demonstrated that in isolated GH-secreting adenomas, as well as in MAS patients with acromegaly, Gsalpha mutations were on the maternal allele. By contrast, the involvement of other endocrine organs in MAS patients was not associated with a particular parent specificity, as precocious puberty and hyperthyroidism were present in patients with mutations on either the maternal or the paternal allele. Moreover, isolated hyperfunctioning thyroid and adrenal adenomas displayed the mutation on the maternal and paternal alleles, respectively. These data confirm the importance of Gsalpha imprinting in the pituitary gland and point out the high degree of tissue specificity of this phenomenon.

In vitro and in vivo studies have shown that oxaliplatin (L-OHP), 5-fluorouracil (5-FU) and leucovorin (L) have a synergistic activity on metastatic colorectal cancer (MCC). In order to better exploit the synergism of action between the three drugs, L-OHP was administered over 2 days, together with 5-FU-L, in a cohort of patients with MCC that had been pre-treated with chemotherapy. Forty-six patients were entered into the trial. All had been pre-treated with chemotherapy for metastatic disease: 14 with the 'de Gramont' regimen alone, and 32 with the same regimen combined with irinotecan (CPT-11). The outpatient treatment consisted of L-OHP 50 mg/m(2), followed immediately by the 'de Gramont' regimen. All drugs were administered on days 1 and 2, every 14 days. Median patient age was 65 years (range: 46-78), male/female ratio was 29/17. All 46 patients were evaluated for response and toxicity. We observed 1 complete response (2.2%) and 14 partial responses (30.4%), giving an overall response rate of 32.6% (95% CI: 19.5-48.06%); 22 patients had stable disease (47.8%) and 9 patients progressed (19.6%). After a median follow-up of 13 months, median time to progression was 6.4 months (range: 3.1-31.2+), while overall median survival was 12.2 months (range: 3.7-31.2+). Toxicity was manageable: grade 3 or 4 neutropenia was observed in 33% of patients, while only 6% of patients had grade 1-2 neurotoxicity. The fractionated bimonthly schedule of L-OHP plus 5-FU-L, showed activity, with an acceptable toxicity profile, both in patients with MCC pre-treated with the 'de Gramont' regimen alone, or with this regimen associated with CPT-11.

Mutations in the guanine nucleotide binding alpha-subunit 1 gene (GNAS1) cause Albright's hereditary osteodistrophy, and the parent of transmission determines variable phenotypic expression of the disease. This has suggested that GNAS1 may be under tissue-specific imprinting control, although studies so far available have failed to clearly define the pattern of GNAS1 expression in humans. To establish if GNAS1 is imprinted in human endocrine tissues, we selected 14 thyroid, 10 granulosa cell, 13 pituitary (3 normal glands, 7 GH-secreting adenomas, and 3 nonfunctioning adenomas), 3 adrenal, and 11 lymphocyte samples shown to be heterozygous for a known polymorphism in exon 5. RNA from these tissues was analyzed by RT-PCR, and expression from both parental alleles was evaluated by enzymatic digestion and subsequent quantification of the resulting fragments. The parental origin of Gs alpha was assessed by evaluating neuroendocrine secretory protein 55 and extra large alphas-like protein transcripts, which have been shown to be monoallelically and parent-specifically expressed from the maternal and paternal allele, respectively. By this approach, the great majority of thyroid (n = 12), ovarian (n = 7), and pituitary (n = 11) samples showed an almost exclusive or significantly predominant expression of the maternal allele over the paternal one, whereas in lymphocyte and adrenal samples both alleles were equally expressed. Our results provide evidence for a predominant maternal origin of GNAS1 transcripts in different human adult endocrine tissues, particularly thyroid, ovary, and pituitary, and strongly suggest that this mechanism may play a crucial role in the determination of the phenotypic expression of Albright's hereditary osteodistrophy.

Departments of Medical Sciences (M.F., U.V., F.E., P.B.-P., G.M., A.G.L., A.S.) and Obstetrics and Gynecology (L.T.), University of Milan, 20122 Milan Italy; Endocrinology and Diabetology Unit (M.F., U.V., F.E., P.B.-P., G.M., A.S.), Medical Genetics Unit (F.N., F.L.), and Neuroradiology Unit (S.A.), Fondazione Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Ca’ Granda Ospedale Maggiore Policlinico, 20122 Milan, Italy; and Endocrine Unit (A.G.L.), Istituto Clinico Humanitas IRCCS, 20089 Rozzano, Milan, Italy

Both cAMP production by adenylyl cyclase and cAMP degradation by phosphodiesterases account for intracellular cAMP levels. We previously demonstrated an increased phosphodiesterase activity in GH-secreting adenomas bearing the gsp oncogene. Here we characterize both the activity and the expression of cAMP-specific phosphodiesterase genes in the human pituitary and in gsp+ and gsp- GH-secreting adenomas and analyze the impact of this intracellular feedback mechanism on the levels of cAMP-responsive element-binding protein phosphorylation. Normal pituitary and gsp- GH-secreting adenomas showed similar phosphodiesterase activities, and 7-fold higher levels were observed in gsp+ tumors. In these tumors the increased activity was mainly owing to isobutyl-methyl-xanthine-sensitive phosphodiesterase 4 and to isobutyl-methyl-xanthine-insensitive isoforms. By semiquantitative RT-PCR, all phosphodiesterase 4 transcripts were expressed in the normal and tumoral pituitary. However, the levels of phosphodiesterase 4C and 4D messenger RNAs were significantly higher in gsp+ than in gsp- GH-secreting adenomas and normal pituitary. Expression of the thyroid-specific isobutyl-methyl-xanthine-insensitive phosphodiesterase 8B was absent in the normal pituitary but detectable in almost all GH-secreting adenomas and higher in gsp+ (P < 0.02). Therefore, this study provides a characterization of phosphodiesterase expression in human pituitary and demonstrates a dramatic induction of the cAMP-specific phosphodiesterases 4C and phosphodiesterases 4D and phosphodiesterases 8B in gsp+ GH-secreting adenomas. Similar levels of cAMP-responsive element-binding protein phosphorylation were observed in gsp- and gsp+ GH-secreting adenomas; however, phosphodiesterase blockade caused an increase in cAMP-responsive element-binding protein phosphorylation that was significantly higher in gsp+ than in gsp- adenomas. Because cAMP-responsive element-binding protein represents the principal end point of the cAMP pathway, these results suggest that the enhanced phosphodiesterase activity may have a significant impact on the phenotypic expression of gsp mutations.

Thyroid transcription factor-1 (TTF-1), a tissue-specific nuclear transcription factor involved in the embryogenesis and differentiation of human thyroid, lung and brain, has been recently identified in other rat tissues, including parafollicular C cells and parathyroid chief cells. Based on this distribution, a possible role for this factor in calcium homeostasis has been suggested. This study investigated the presence of TTF-1 transcripts and protein in human tissues expressing the calcium sensing receptor (CaSR). Using a RT-PCR technique, complemented by Southern blot analysis, TTF-1 expression was detected in human C cells (two medullary thyroid carcinomas), but not in normal and adenomatous (four adenomas and three hyperplasia) parathyroid, and normal and adenomatous (six adenomas) pituitary tissues. CaSR was expressed in all samples. The absence of expression was confirmed by Western blot. In contrast to previous studies in the rat, this study demonstrates the absence of TTF-1 transcripts in the human adult parathyroid and pituitary glands, although a role for this factor during the ontogeny of these organs cannot be excluded.

1.5 Mutational spectrum In E70–80% of cases, PHP-Ia is caused by haploinsufficiency because of maternally-inherited heterozygous inactivating mutations of GNAS gene1,2 (personal data). In very few cases, large deletions including part or the whole gene have been reported,3,4 while in a further subset of patients cytosine methylation defects of the GNAS promoters have been identified, a pattern typically found in PHP-Ib.5–8 Patients affected with PHP-Ib share a loss of methylation on the maternal A/B exon of GNAS. Those with the autosomal dominant form of PHP-Ib (AD-PHP-Ib) display an isolated loss of methylation at exon A/B associated with a recurrent 3-kb deletion in the STX16 gene, although a 4.4-kb deletion has been described in one family with AD-PHP-Ib.9,10 In four families with AD-PHP-Ib, NESP55 and NESPAS deletions have also been described leading to the loss of all maternal GNAS imprints (epimutations).11–13 In most cases, PHP-Ib is sporadic, and is characterized by complete loss of methylation at the NESPas, XLas and A/B promoters, and no other changes in cisor trans-acting elements have been found to explain this loss of methylation. In some cases (2–20% of the patients with PHP-Ib) paternal 20q disomies have been described.14–17