Sergio Ferasin

A Novel Mutation in the Upstream Open Reading Frame of the CDKN1B Gene Causes a MEN4 Phenotype

The CDKN1B gene encodes the cyclin-dependent kinase inhibitor p27KIP1, an atypical tumor suppressor playing a key role in cell cycle regulation, cell proliferation, and differentiation. Impaired p27KIP1 expression and/or localization are often observed in tumor cells, further confirming its central role in regulating the cell cycle. Recently, germline mutations in CDKN1B have been associated with the inherited multiple endocrine neoplasia syndrome type 4, an autosomal dominant syndrome characterized by varying combinations of tumors affecting at least two endocrine organs. In this study we identified a 4-bp deletion in a highly conserved regulatory upstream ORF (uORF) in the 5′UTR of the CDKN1B gene in a patient with a pituitary adenoma and a well-differentiated pancreatic neoplasm. This deletion causes the shift of the uORF termination codon with the consequent lengthening of the uORF–encoded peptide and the drastic shortening of the intercistronic space. Our data on the immunohistochemical analysis of the patients pancreatic lesion, functional studies based on dual-luciferase assays, site-directed mutagenesis, and on polysome profiling show a negative influence of this deletion on the translation reinitiation at the CDKN1B starting site, with a consequent reduction in p27KIP1 expression. Our findings demonstrate that, in addition to the previously described mechanisms leading to reduced p27KIP1 activity, such as degradation via the ubiquitin/proteasome pathway or non-covalent sequestration, p27KIP1 activity can also be modulated by an uORF and mutations affecting uORF could change p27KIP1 expression. This study adds the CDKN1B gene to the short list of genes for which mutations that either create, delete, or severely modify their regulatory uORFs have been associated with human diseases.

Performance of salivary cortisol in the diagnosis of Cushing's syndrome, adrenal incidentaloma, and adrenal insufficiency.

OBJECTIVE Salivary cortisol has recently been suggested for studies on the hypothalamic-pituitary-adrenal (HPA) axis. The lack of circadian rhythm is a marker of Cushings syndrome (CS), and some authors have reported that low salivary cortisol levels may be a marker of adrenal insufficiency. The aim of our study was to define the role of salivary cortisol in specific diagnostic settings of HPA axis disease. SUBJECTS AND METHODS We analyzed morning salivary cortisol (MSC) and late-night salivary CORTISOL (LNSC) levels in 406 SUBJECTS: 52 patients with Cushings disease (CD), 13 with ectopic CS, 17 with adrenal CS, 27 with CD in remission (a mean follow-up of 66±39 months), 45 with adrenal incidentaloma, 73 assessed as having CS and then ruled out for endogenous hypercortisolism, 75 with adrenal insufficiency, and 104 healthy subjects. RESULTS A LNSC value above 5.24  ng/ml differentiated CS patients from controls with high sensitivity (96.3%) and specificity (97.1%); we found higher LNSC levels in ectopic CS patients than in CD patients. We found no difference in MSC and LNSC levels between patients with CD in remission and healthy subjects. Both MSC and LNSC levels were higher in patients with adrenal incidentaloma than in healthy controls. A MSC value below 2.65 ng/ml distinguished patients with adrenal insufficiency from controls with high sensitivity (97.1%) and specificity (93.3%). CONCLUSIONS Salivary cortisol is a useful tool to assess endogenous cortisol excess or adrenal insufficiency and to evaluate stable CD in remission.

Assessment of glucocorticoid therapy with salivary cortisol in secondary adrenal insufficiency

CONTEXT Appropriate glucocorticoid replacement therapy in adrenal insufficiency (AI) is crucial, given the risks of chronic under- or overtreatment, particularly in patients on multiple medications. Salivary sampling allows for non-invasive, stress-free cortisol measurement. OBJECTIVE To determine whether salivary cortisol measurement is helpful in assessing the adequacy of glucocorticoid therapy with cortisone acetate (CA) in patients with secondary AI. DESIGN A prospective cohort study at the Endocrinology Unit of Padua University Hospital. METHODS Six samples of salivary cortisol were collected from 28 patients with secondary AI on CA treatment and from 36 healthy volunteers at fixed times of the day, and used to calculate salivary cortisol levels at each time point and the area under the curve (AUC) across the different sampling times. RESULTS Salivary cortisol levels were lower in patients than in controls in the morning but no differences were found in the afternoon or at night before resting. Salivary cortisol levels were higher in patients immediately following CA administration. Ten patients showed an AUC above the 97.5th percentile of controls, without clinical signs of hypercortisolism, and salivary cortisol levels 90 min after each dose of CA predict the AUC. All patients had severe GH deficiency and there were no differences in salivary cortisol levels or AUC between patients treated or not with GH. CONCLUSIONS Two salivary cortisol determinations, able to predict the daily AUC, may allow for assessing the adequacy of glucocorticoid replacement therapy in secondary AI and for identifying cases of over- or undertreatment.

Activation of the Dopamine Receptor Type-2 (DRD2) Promoter by 9-Cis Retinoic Acid in a Cellular Model of Cushing's Disease Mediates the Inhibition of Cell Proliferation and ACTH Secretion Without a Complete Corticotroph-to-Melanotroph Transdifferentiation

Cushings disease (CD) is a rare condition in which hypercortisolemia is secondary to excessive ACTH release from a pituitary corticotroph adenoma. CD is associated with significant morbidity and mortality, and a safe therapy that effectively targets the pituitary tumor is still lacking. Retinoic acid (RA) and dopamine agonists (DAs) have recently been considered as monotherapy in CD patients, and satisfactory results have been reported, albeit in a limited number of patients. Given the permissive role of RA on the dopamine receptor type-2 (DRD2), the aim of the present study was to see whether a combination of 9-cis RA and the DA bromocriptine (Br) might represent a possible treatment for CD. Here we show that 9-cis RA induces a functional DRD2 in the pituitary corticotroph cell line AtT20, and increases cell sensitivity to Br via a mechanism only partially related to corticotroph-to-melanotroph transdifferentiation. In addition, 9-cis RA and Br act synergistically to modulate cell viability, with favorable implications for clinical use. In nearly 45% of corticotropinoma-derived primary cultures, the combined administration of 9-cis RA and Br lowered the steady-state level of the ACTH precursor proopiomelanocortin (POMC) more efficiently than either of the drugs alone. In conclusion, the effects of a combination of 9-cis RA and Br on ACTH synthesis/secretion and cell viability in AtT20, and on POMC transcriptional activity in human corticotropinomas might represent a suitable starting point for assessing the potential of this treatment regimen for ACTH-secreting pituitary adenomas. This study thus has potentially important implications for novel therapeutic approaches to CD.

The Glucose‐Dependent Insulinotropic Polypeptide Receptor is Overexpressed Amongst GNAS1 Mutation‐Negative Somatotropinomas and Drives Growth Hormone (GH)‐Promoter Activity in GH3 Cells

Somatic mutations in the GNAS1 gene, encoding the α‐subunit of the heterotrimeric stimulatory G protein (Gαs), occur in approximately 40% of growth hormone (GH)‐secreting pituitary tumours. By altering the adenylate cyclase‐cAMP‐protein kinase A pathway, they unequivocally give somatotroph cells a growth advantage. Hence, the pathogenesis of somatotropinomas could be linked to anomalies in receptors coupled to the cAMP second‐messenger cascade. Among them, the glucose‐dependent insulinotropic polypeptide receptor (GIPR) is already known to play a primary role in the impaired cAMP‐dependent cortisol secretion in patients affected by food‐dependent Cushing’s syndrome. In the present study, 43 somatotropinomas and 12 normal pituitary glands were investigated for GIPR expression by quantitative reverse transcriptase‐polymerase chain reaction, western blotting and immunohistochemistry. Tumoural specimens were also evaluated for GNAS1 mutational status. The effect of GIPR overexpression on cAMP levels and GH transcription was evaluated in an in vitro model of somatotropinomas, the GH‐secreting pituitary cell line GH3. GIPR was expressed at higher levels compared to normal pituitaries in 13 GNAS1 mutation‐negative somatotropinomas. GIP stimulated adenylyl cyclase and GH‐promoter activity in GIPR‐transfected GH3 cells, confirming a correct coupling of GIPR to Gαs. In a proportion of acromegalic patients, GIPR overexpression appeared to be associated with a paradoxical increase in GH after an oral glucose tolerance test. Whether GIPR overexpression in acromegalic patients may be associated with this paradoxical response or more generally involved in the pathogenesis of acromegaly, as suggested by the mutually exclusive high GIPR levels and GNAS1 mutations, remains an open question.

Age and the metabolic syndrome affect salivary cortisol rhythm: data from a community sample.

OBJECTIVEMeasurement of Cortisol levels in saliva is a marker of free hormone. How salivary Cortisol rhythm is affected by age, gender, the metabolic syndrome and estrogen-progestin therapy was evaluated in a community sample of adults.SUBJECTS AND METHODSOne hundred twenty volunteers recruited from the Hospital staff and family members of the Endocrinology Unit were instructed to collect 7 salivary samples: the first on awakening (F0) and 6 more (F1.5, F5, F6, F10, F11.5 and F14) over the next 14 hours. Each volunteer also underwent a complete physical evaluation and a comprehensive medical history was taken. Salivary Cortisol was measured using a radioimmunometric assay. Daily Cortisol secretion was evaluated computing the Area Under the Curve (AUCF0→F14); the F14/F0 ratio was calculated as a marker of Cortisol rhythm.RESULTSMedian F14 levels were higher in the subjects in the third tertile of age than in those falling in the second or in the first age tertile (respectively, 2.09 vs 1.33 vs 1.25 ng/mL, p=0.023 and p=0.006), in the hypertensive volunteers (2.44 vs 1.44 ng/mL, p=0.030) and in those with the metabolic syndrome (2.95 vs 1.4 ng/mL, p=0.002), with an elevated median F14/F0 ratio (0.48 vs 0.19, p=0.006). According to the Kruskal-Wallis analysis of variance, the most important factor affecting F14 value was age (p=0.001). AUCF0→F14 was not influenced by gender, age, metabolic syndrome or estrogen-progestin therapy.CONCLUSIONSWhile it did not affect the daily Cortisol rate, late-night salivary cortisol levels were found to be increased in the subjects in the higher age tertile and in those with the metabolic syndrome.

Adrenal lesions in acromegaly: Do metabolic aspects and aryl hydrocarbon receptor interacting protein gene have a role? Evaluation at baseline and after long-term follow-up

Background: Adrenal lesions are discovered in acromegaly more frequently than in general population, without relationship with primary disease. Some patients, carriers of aryl hydrocarbon receptor interacting protein (AIP) gene mutations, developed an adrenal neoplasm. Aim: To evaluate the role of metabolic and genetic aspects and the follow-up of adrenal nodules in acromegaly. Material and methods: We studied 69 acromegalic patients (30 male and 39 female, 56±15 yr) who had been referred to the Endocrinology Unit of Padua. In all patients we determined body mass index (BMI) and waist-to-hip ratio (WHR); we performed an oral glucose tolerance test (OGTT) whenever possible. If adrenal computed tomography revealed a lesion, the patient underwent an endocrine and genetic study. Results: Adrenal lesions were identified in 14 patients and were not related to gender, duration of disease, GH or IGF-I concentrations, basal and after-OGTT glucose and insulin levels, log(HOMA-IR) and Quantitative Insulin Sensitivity Check Index (QUICKI) values, whereas BMI and WHR were higher in patients with adrenal lesions. Baseline endocrine and radiological study revealed benign lesions; during mean 4-yr follow-up none of the patients showed hormone excess, even though some lesions increased in size. We did not find any mutation in AIP gene, except heterozygous silent alteration (T48T). Conclusions: The frequency of non-functioning adrenal lesions in acromegaly is not associated with the considered aspects, except BMI and WHR. The prolonged follow-up showed that these lesions have a tendency to increase in size independently of the control of acromegaly, so a morphological follow-up is recommended.

Adrenal nodules in patients with Cushing’s disease: Prevalence, clinical significance and follow-up

Adrenal glands in Cushing’s disease (CD) range from normal to showing diffuse enlargement in most cases. The finding of nodular lesions has been reported, but information about prevalence and evolution is described in few reports. Aim: To investigate the prevalence of nodular adrenal glands in patients with CD and assess its evolution after disease remission. Subjects and methods: We assessed 41 CD patients’ abdominal computed tomography (CT) scans obtained during the active phase of the disease and evaluated the dynamics of ACTH and cortisol secretion. CT was repeated after disease remission in patients with adrenal nodules. Results: Fifteen of 41 patients had nodular and the remaining 26 had normal or enlarged adrenal glands. Patients with nodules were older (45.1 ±8.8 vs 36.9±12.7 yr; p=0.03) and had longer-standing disease (57.3±56.9 vs 32.9±29.1 months; p=0.05) than patients with normal/enlarged adrenal glands. ACTH (45.4±21.3 vs 70.5±39.1 pg/ml; p=0.04) and urinary free cortisol levels (606.1 ±512.3 vs 301.0±224.7 µg/day, p=0.01) were significantly lower in patients with adrenal nodules while there were no differences between the groups in terms of dynamic tests results. Post-operative follow-up showed regression or shrinkage of the nodules in 8 out of 10 patients in disease remission. Conclusions: We found that adrenal nodular glands are a frequent finding in CD in particular in older patients and in those with a longer-standing disease. Nevertheless, a high percentage of nodules regression or shrinking was evidenced in our series after disease remission.