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

PubMed ID: 23555276This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

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

Abstract OBJECTIVE: Measurement 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 METHODS: One 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 (F(0)) and 6 more (F(1.5), F(5), F(6), F(10), F(11.5) and F(14)) 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 (AUC(F0)(→)(F14)); the F(14)/F(0) ratio was calculated as a marker of cortisol rhythm. RESULTS: Median F(14) 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 F(14)/F(0) ratio (0.48 vs 0.19, p=0.006). According to the Kruskal-Wallis analysis of variance, the most important factor affecting F(14) value was age (p=0.001). AUC(F0)(→)(F14) was not influenced by gender, age, metabolic syndrome or estrogen-progestin therapy. CONCLUSIONS: While 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 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 mu g/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

Assessment of glucocorticoid therapy with salivary cortisol in secondary adrenal insufficiency.

CONTEXT: Appropriate glucocorticoid replacement therapy in adrenal insufficiency (AI) is crucial, given to the risks of chronic under- or over treatment, particularly in patients on multiple medications. Salivary sampling allows 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 Curve (AUC) across the different sampling times. RESULTS: Salivary cortisol levels were lower in patients than 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 minutes after each dose of CA predict the AUC. All patients had severe GH-deficiency: 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 assessment of the adequacy of glucocorticoid replacement therapy in secondary AI and for identifying cases of over- or under-treatment

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

Salivary cortisol has been recently suggested for studies on the hypothalamic-pituitary-adrenal (HPA) axis: lack of circadian rhythm is a marker of Cushing's syndrome (CS), and some authors report 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 setting of HPA axis disease. SUBJECTS AND METHODS: We analyzed morning salivary cortisol (MSC) and late night salivary cortisol (LNSC) in 406 subjects: 52 Cushing's disease (CD), 13 ectopic-CS, 17 adrenal-CS, 27 CD in remission (mean follow-up of 66 \ub1 39 months), 45 adrenal incidentalomas, 73 patients assessed of CS and then ruled out for endogenous hypercortisolism, 75 patients with adrenal insufficiency and 104 healthy subjects. RESULTS: A LNSC value above 5.24 ng/mL differentiated CS from controls with high sensitivity (96.3%) and specificity (97.1%), we found higher LNSC in ectopic-CS than in CD. We found no difference in MSC and LNSC levels between CD in remission and healthy subjects. Both MSC and LNSC were higher in adrenal incidentaloma than in healthy controls. MSC 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

Venous thromboembolism in patients with Cushing's syndrome: need of a careful investigation of the prothrombotic risk profile.

A high incidence of venous thromboembolic (VTE) complications has been reported in Cushing's syndrome (CS), mostly post-operatively and attributable to hypercoagulability. The prevalence of symptomatic VTE was investigated retrospectively in 58 consecutive CS patients in relation to acquired and genetic thrombotic risk factors. Eight CS patients (14 %) developed VTE (group A), 3 of them related and 5 unrelated to surgery. These patients had higher urinary free cortisol (p = 0.01) and VWF levels (p = 0.02) than the 50 patients without VTE (group B), as well an increase in the hemostatically more efficient, high-molecular-weight VWF multimers (p = 0.002). Factor V Leiden and the prothrombin gene 20210A variants (the most common inherited thrombophilic defects) were more represented in group A than in group B, as was the genotype GCAG/GCAG of the VWF gene promoter, known to hyperinduce VWF upregulation under cortisol excess. All but one of the patients with VTE unrelated to surgery had at least four acquired and at least one inherited risk factor. Severe hypercortisolism and VWF levels with increased haemostatic activity are strongly associated with VTE in CS. VTE episodes unrelated to surgery are attributable to the synergistic action of acquired and inherited thrombotic risk factors. Based on these observations, we believe that severely affected CS patients should be screened for coagulation disorders and receive antithrombotic prophylaxis whenever they have concomitant prothrombotic risk factors

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

Cushing\u2019s 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 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 Cushing\u2019s disease

Constructs used in transfection experiments.

<p>(A) Either the wild type, the c.-456_-453delCCTT or the c.-469C>T containing 5′UTRs were cloned upstream the firefly luciferase gene. (B) The wild type/c.-456_-453delCCTT 5′UTR were subcloned upstream the <i>CDKN1B</i> gene. (C) The introduction of the c.-428A>T substitution in the two former plasmids reported in panel A restores uORF length and intercistonic distance. (D) Constructs reported in panel B were mutated introducing a c.-74insC leading to the translation of a chimeric protein in the double mutant.</p

Polysome profiling of lymphoblastoid cells from the -456_-453delCCTT mutation carrier.

<p>(A) Representative absorbance profile for RNA separated by velocity sedimentation through a 15–50% sucrose gradient. The positions of the 40S, 60S, 80S, and polysomal peaks are indicated. (B) The levels of <i>CDKN1B</i> mRNA (wt and c.-456_-453del) in each gradient fraction were measured by quantitative real-time PCR and plotted as a percentage of the total <i>CDKN1B</i> mRNA levels (wt or c.-456_-453del, respectively) in that sample. Data represent three independent experiments, with mean ± SD reported. (C) The levels of both alleles in each fraction were expressed as relative quantities calculated from differences in Cq values between mRNA and genomic DNA for removing the intrinsic variation between the two qPCR assays. Data represent the mean of three independent experiments ± SD.</p