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Elisa PIGNATTI

Personale tecnico amministrativo
Dipartimento Chirurgico, Medico, Odontoiatrico e di Scienze Morfologiche con interesse Trapiantologico, Oncologico e di Medicina Rigenerativa

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Pubblicazioni

2024 - A comprehensive review on the role of mesenchymal stromal/stem cells in the management of rheumatoid arthritis [Articolo su rivista]
Pignatti, Elisa; Maccaferri, Monia; Pisciotta, Alessandra; Carnevale, Gianluca; Salvarani, Carlo
abstract

Introduction: Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease with systemic manifestations. Although the success of immune modulatory drug therapy is considerable, about 40% of patients do not respond to treatment. Mesenchymal stromal/stem cells (MSCs) have been demonstrated to have therapeutic potential for inflammatory diseases. Areas covered: This review provides an update on RA disease and on pre-clinical and clinical studies using MSCs from bone marrow, umbilical cord, adipose tissue, and dental pulp, to regulate the immune response. Moreover, the clinical use, safety, limitations, and future perspective of MSCs in RA are discussed. Using the PubMed database and ClincalTrials.gov, peer-reviewed full-text papers, abstracts and clinical trials were identified from 1985 through to April 2023. Expert opinion: MSCs demonstrated a satisfactory safety profile and potential for clinical efficacy. However, it is mandatory to deepen the investigations on how MSCs affect the proinflammatory deregulated RA patients' cells. MSCs are potentially good candidates for severe RA patients not responding to conventional therapies but a long-term follow-up after stem cells treatment and standardized protocols are needed. Future research should focus on well-designed multicenter randomized clinical trials with adequate sample sizes and properly selected patients satisfying RA criteria for a valid efficacy evaluation.

2023 - Assessing the Ability of Human Dental Pulp Stem Cells to Modulate the Macrophages Phenotype. [Poster]
Maccaferri, M; Pisciotta, A; Carnevale, G; Salvarani, C; Pignatti, E.
abstract

2023 - Checkpoint immunitari sulle cellule staminali della polpa dentale umana modulati in vitro da linfociti e monociti da pazienti con artrite reumatoide [Poster]
Rossi, Alessandro; Bonacini, Martina; Ferrigno, Ilaria; Carnevale, Gianluca; Pisciotta, Alessandra; DI TINCO, Rosanna; Pignatti, Elisa; Catanoso, Mariagrazia; Crescentini, Filippo; Citriniti, Giorgia; Magnani, Luca; Caruso, Andrea; Germanò, Giuseppe; Brandolino, Fabio; Chiapparoli, Ilaria; Dardani, Lucia; Cocchiara, Emanuele; Zerbini, Alessandro; Salvarani, Carlo; Croci, Stefania
abstract

2023 - Effect of the Inflammatory Microenvironment Induced by Monocytes on Fibroblasts and Modulatory Action of Human Dental Pulp Stem Cells. [Poster]
Maccaferri, M; Pisciotta, A; Carnevale, G; Salvarani, C; Pignatti, E.
abstract

2023 - EFFETTO DEL MICROAMBIENTE INFIAMMATORIO INDOTTO DAI MONOCITI SUI FIBROBLASTI E AZIONE MODULATORIA DELLE CELLULE STAMINALI DELLA POLPA DENTALE UMANA [Poster]
Maccaferri, Monia; Corbelli, Tommaso; Lazzari, Giorgia; Pisciotta, Alessandra; Carnevale, Gianluca; Salvarani, Carlo; Pignatti, Elisa
abstract

2023 - Human dental pulp stem cells (hDPSCs) promote the lipofibroblast transition in the early stage of a fibro-inflammatory process [Articolo su rivista]
Pisciotta, Alessandra; DI TINCO, Rosanna; Bertani, Giulia; Orlandi, Giulia; Bertoni, Laura; Pignatti, Elisa; Orciani, Monia; Sena, Paola; Bertacchini, Jessika; Salvarani, Carlo; Carnevale, Gianluca
abstract

Introduction: In autoimmune diseases, particularly in systemic sclerosis and chronic periaortitis, a strict correlation between chronic inflammation and fibrosis exists. Since the currently used drugs prove mostly effective in suppressing inflammation, a better comprehension of the molecular mechanisms exerted by cell types implicated in fibro-inflammation is needed to develop novel therapeutic strategies. Mesenchymal stromal/stem cells (MSCs) are being matter of deep investigation to unveil their role in the evolution of fibrogenetic process. Several findings pointed out the controversial implication of MSCs in these events, with reports lining at a beneficial effect exerted by external MSCs and others highlighting a direct contribution of resident MSCs in fibrosis progression. Human dental pulp stem cells (hDPSCs) have demonstrated to hold promise as potential therapeutic tools due to their immunomodulatory properties, which strongly support their contribution to tissue regeneration. Methods: Our present study evaluated hDPSCs response to a fibro-inflammatory microenvironment, mimicked in vitro by a transwell co-culture system with human dermal fibroblasts, at early and late culture passages, in presence of TGF-β1, a master promoter of fibrogenesis. Results and Discussion: We observed that hDPSCs, exposed to acute fibro-inflammatory stimuli, promote a myofibroblast-to-lipofibroblast transition, likely based on BMP2 dependent pathways. Conversely, when a chronic fibro-inflammatory microenvironment is generated, hDPSCs reduce their anti-fibrotic effect and acquire a pro-fibrotic phenotype. These data provide the basis for further investigations on the response of hDPSCs to varying fibro-inflammatory conditions.

2023 - Reversibility in male idiopathic osteoporosis possible [Articolo su rivista]
Jamall, Ijaz S; Ullery, Michael C; Rocchietti March, Massimiliano; Pignatti, Elisa; Rochira, Vincenzo; Brücher, Björn L D M
abstract

Summary: A 44-year-old athletic man presented in 2009 with severe low back pain. Dual-energy x-ray absorptiometry revealed severe osteoporosis; serum testosterone was 189 ng/dL while serum estradiol (E2) measured by liquid chromatography/mass spectrometry was 8 pg/mL. DNA was extracted and sequenced from a blood sample from the patient since his maternal first cousin also had low bone mass and both patients were screened for aromatase dysfunction by PCR analysis for the CYP19A1 gene, which encodes aromatase. No known pathologic mutations were observed in the coding exons, but novel single nucleotide polymorphisms were detected both in the proband and in his cousin. Treatment with topical testosterone started in August 2010. Over the next 8 years, testosterone dosage was varied and switched from topical gel to injections and maintained on depo-injections of testosterone at about 60 mg once per week. Re-examination in March 2012 included a brain MRI to exclude pituitary lesions; hyperparathyroidism was ruled out (normal serum parathyroid hormone, calcium, and calcium to phosphorous ratio) and celiac disease was excluded (negative transglutaminase antibodies). Follow-up in October 2018 showed improved bone mineral density of the lumbar spine by 29% and of the left femoral hip by 15% compared to baseline measurements. This reveals the importance of measuring serum E2 for making the correct diagnosis, as well as for monitoring a therapeutic effect. Herein, we propose treatment of male osteoporosis where serum E2 levels are below about 20 pg/mL with testosterone to reverse osteoporosis. Learning points: Estrogen deficiency in the diagnosis of male idiopathic osteoporosis. Importance of serum estradiol in male osteoporosis. Role of polymorphisms in aromatase gene on bone health. Reversal of osteoporosis. Tailored testosterone treatment for bone health.

2023 - Studio in vitro dei tempi di risposta dei sinoviociti di tipo fibroblastico allo stimolo infiammatorio [Poster]
Maccaferri, M.; Corbelli, T.; Lazzari, G.; Salvarani, C.; Pignatti, E.
abstract

2022 - MODULAZIONE DEI MONOCITI NEI PROCESSI INFIAMMATORI CON IL CONTRIBUTO DELLE CELLULE STAMINALI DELLA POLPA DENTALE UMANA (hDPSC) [Poster]
Pignatti, E.; Maccaferri, M.; Pisciotta, A.; Di Tinco, R.; Bertani, G.; Bertoni, L.; Croci, S.; Bonacini, M.; Carnevale, G.; Salvarani, C.
abstract

2022 - RUOLO DEI MACROFAGI IN CONDIZIONI PRO- ED ANTI-INFIAMMATORIE E MECCANISMI DI REGOLAZIONE INDOTTI DALLE hDPSCs [Poster]
Maccaferri, M.; Pisciotta, A.; Di Tinco, R.; Bertani, G.; Bertoni, L.; Carnevale, G.; Salvarani, C.; Pignatti, E.
abstract

2021 - Human Dental Pulp Stem Cells Modulate Cytokine Production in vitro by Peripheral Blood Mononuclear Cells From Coronavirus Disease 2019 Patients [Articolo su rivista]
Croci, S.; Bonacini, M.; Dolci, G.; Massari, M.; Facciolongo, N.; Pignatti, E.; Pisciotta, A.; Carnevale, G.; Negro, A.; Cassone, G.; Muratore, F.; Belloni, L.; Zerbini, A.; Salvarani, C.
abstract

A subset of patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) developed a condition of hyper-inflammation, which can cause multi-organ damage and the more severe forms of coronavirus disease 2019 (COVID-19). Mesenchymal stem cells (MSCs) can promote tissue regeneration and modulate immune responses and, thus, have the rational requirements to be used to counteract SARS-CoV-2-induced pneumonia and hyper-inflammation. The aim of the present study was to gain insight into possible mechanisms of action of MSCs obtained from human dental pulp [dental pulp stem cells (DPSCs)] in COVID-19 patients. We investigated the concentrations of 18 cytokines in supernatants of peripheral blood mononuclear cells (PBMCs) obtained from COVID-19 patients cultured in vitro alone and in contact with DPSCs. The modulation of cytokines in PBMCs was confirmed by real-time PCR. IL-6 was the sole cytokine detected in supernatants of DPSCs. In resting conditions, co-culture increased IL-1β, IL-2, IL-5, IL-6, IL-10, IL-18, TNFα, and granulocyte macrophage colony-stimulating factor (GM-CSF) levels. When PBMCs were activated with anti-CD3/CD28 antibody-coated beads, co-culture increased IL-6 and GM-CSF, whereas it decreased IFNγ, TNFα, IL-2, IL-5, IL-9, IL-10, IL-12 (p70), IL-17A, IL-18, IL-21, IL-23, and IL-27 levels. Concentrations of IL-1β, IL-4, IL-13, and IL-22 were not affected. The comparison of cytokine concentrations in supernatants of PBMCs from COVID-19 patients vs. healthy subjects revealed lower concentrations of IL-10 and higher concentrations of IL-18 in supernatants of CD3/CD28-activated PBMCs from COVID-19 patients. Results are explorative but indicate that DPSCs can modulate the production of cytokines deregulated in COVID-19 patients, supporting their potential use in COVID-19.

2021 - Real-life use of BRAF-V600E mutation analysis in thyroid nodule fine needle aspiration: consequences on clinical decision-making [Articolo su rivista]
Brigante, G.; Craparo, A.; Pignatti, E.; Marino, M.; Monzani, M. L.; De Vincentis, S.; Casarini, L.; Sperduti, S.; Boselli, G.; Margiotta, G.; Ippolito, M.; Rochira, V.; Simoni, M.
abstract

Purpose: This study aimed to evaluate the real-life use of BRAF-V600E mutation analysis in washout liquid from thyroid nodule fine needle aspiration (FNA), and the consequences of genetic result on clinical decision-making. Methods: We retrospectively considered subjects tested for BRAF-V600E among those attending the Endocrinology Unit of Modena for FNA between 2014 and 2018. Washing fluid was collected together with cytological sample and stored at −20 °C. If the clinician deemed it necessary, the sample was thawed, DNA extracted, and genetic test performed by high-resolution melting technique. We collected data on cytology according to the Italian Consensus for the cytological classification of thyroid nodules, type of surgery (when performed), histology, and adverse events. Results: Out of 7112 subjects submitted to FNA, BRAF analysis was requested for 683 (9.6%). Overall, 896 nodules were analyzed: 74% were indeterminate at cytology, mainly TIR3A (low risk). Twenty-two nodules were mutant (BRAF+). Only 2% of indeterminate, mainly TIR3B, were BRAF+. Based on final histological diagnosis, BRAF test had high specificity (100%) but poor sensitivity (21%), also in indeterminate nodules. Mutant subjects underwent more extensive surgery compared to wild type (p = 0.000), with frequent prophylactic central lymph node dissection. One third had local metastases. Higher prevalence of hypoparathyroidism was found in BRAF+ compared to wild type (p = 0.018). Conclusions: The analysis of BRAF-V600E outside of gene panels has low sensitivity, especially in indeterminate nodules, and a positive result could lead to more extensive surgery with greater risk of hypoparathyroidism and questionable clinical utility.

2021 - Role of PD-L1 in licensing immunoregulatory function of dental pulp mesenchymal stem cells [Articolo su rivista]
Di Tinco, R.; Bertani, G.; Pisciotta, A.; Bertoni, L.; Pignatti, E.; Maccaferri, M.; Bertacchini, J.; Sena, P.; Vallarola, A.; Tupler, R.; Croci, S.; Bonacini, M.; Salvarani, C.; Carnevale, G.
abstract

Background: Dental pulp stem cells (DPSCs) are low immunogenic and hold immunomodulatory properties that, along with their well-established multi-potency, might enhance their potential application in autoimmune and inflammatory diseases. The present study focused on the ability of DPSCs to modulate the inflammatory microenvironment through PD1/PD-L1 pathway. Methods: Inflammatory microenvironment was created in vitro by the activation of T cells isolated from healthy donors and rheumatoid arthritis (RA) patients with anti-CD3 and anti-CD28 antibodies. Direct and indirect co-cultures between DPSCs and PBMCs were carried out to evaluate the activation of immunomodulatory checkpoints in DPSCs and the inflammatory pattern in PBMCs. Results: Our data suggest that the inflammatory stimuli trigger DPSCs immunoregulatory functions that can be exerted by both direct and indirect contact. As demonstrated by using a selective PD-L1 inhibitor, DPSCs were able to activate compensatory pathways targeting to orchestrate the inflammatory process by modulating pro-inflammatory cytokines in pre-activated T lymphocytes. The involvement of PD-L1 mechanism was also observed in autologous inflammatory status (pulpitis) and after direct exposure to pre-activated T cells from RA patients suggesting that immunomodulatory/anti-inflammatory properties are strictly related to their stemness status. Conclusions: Our findings point out that the communication with the inflammatory microenvironment is essential in licensing their immunomodulatory properties.

2020 - Clinical practice survey on BRAF V600E role in the therapeutic deci- sion in indeterminate thyroid cytology [Abstract in Atti di Convegno]
Brigante, G.; Craparo, A.; Pignatti, E.; Marino, M.; Casarini, L.; Sperduti, S.; Boselli, G.; Margiotta, G.; Rochira, V.; Simoni, M.
abstract

Introduction The use of multigene panels in thyroid nodule diagnosis is still limited, due to high costs and need for ad hoc sampling. Since BRAF-V600E is the com- monest genetic alteration in differentiated thyroid cancer, this is the mostly tested genetic parameter in clinical practice. Aim To evaluate the use of BRAF mutation analysis in wash-out liquid from fine needle aspiration (FNA) in clinical practice, characterizing the cases in which it is requested, and the consequences of genetic test result on thera- peutic decisions. Methods We considered all the subjects tested for BRAF-V600E among those attend- ing the Endocrinology Unit of Modena for FNA between January 2014 and November 2018. After written informed consent, washing fluid was collected together with cytological sample and stored at –20°C. If the clinician deemed it necessary, the sample was thawed, DNA was extracted and genetic test was performed by the high-resolution melting protocol previously described1. We collected cytology of nodules according to the 2010 SIAPEC-IAP Italian Consensus, and when surgical treatment was performed, histology. Results Out of a total of 7112 subjects submitted to FNA, BRAF analysis was re- quested for 681 (9.6%), for a total of 898 nodules: 97% of nodules were indeterminate at cytology, mainly TIR3A (low risk); 2% suspicious or di- agnostic for cancer, and genetic test was requested to estimate prognosis; 1% were suspect nodules at ultrasonography with unsuspicious cytology. Only 22 nodules were mutant (BRAF+).Most of them were already high risk or suspicious lesions at cytology (64%). One third were TIR3A. Con- sidering the prevalence of BRAF mutation among cytological classes of the whole group, only 1% of TIR3A were BRAF+. Twenty BRAF+ patients were addressed to surgery (one lost at follow-up, one refused): 5% underwent hemithyroidectomy, 25% total thyroidectomy and 70% total thyroidectomy plus central lymph nodes dissection. They all had papillary thyroid cancer. Since 64% of BRAF+ were TIR3B-4-5 at cytology, they had surgical indica- tion even before the genetic test. Among the 14 subjects treated with central neck dissection, only 2 had suspect metastasis before surgery; among those who would have had no indication, one third had metastases (only 1 among TIR3A and 2 among TIR3B). Conclusions Despite the development of panels, single gene tests are still requested, mainly for nodules with indeterminate low risk cytology. BRAF mutation in TIR3A is rare and leads clinicians to more invasive surgery, with question- able clinical utility.

2020 - Modulation of Cell Death and Promotion of Chondrogenic Differentiation by Fas/FasL in Human Dental Pulp Stem Cells (hDPSCs) [Articolo su rivista]
Pisciotta, Alessandra; Bertani, Giulia; Bertoni, Laura; Di Tinco, Rosanna; De Biasi, Sara; Vallarola, Antonio; Pignatti, Elisa; Tupler, Rossella; Salvarani, Carlo; de Pol, Anto; Carnevale, Gianluca
abstract

2019 - Expression and clinicopathological role of miR146a in thyroid follicular carcinoma. [Articolo su rivista]
Pignatti, E.; Vighi, E.; Magnani, E.; Kara, E.; Roncati, L.; Maiorana, A.; Santi, D.; Madeo, B.; Cioni, K.; Carani, C.; Rochira, V.; Simoni, M.; Brigante, Giulia
abstract

PURPOSE: Dysregulation of microRNA expression has been involved in the development and progression of follicular thyroid carcinoma (FTC). The aim of this work was to study the expression of miRNA146a in FTC and the association with clinicopathological features of the disease. METHODS: Thirty-eight patients affected by FTC were included in the study. Twenty patients carrying follicular thyroid adenoma (FA) were also enroled as the benign counterpart of FTC. Total RNA including miRNA146a was extracted from formalin-fixed paraffin-embedded (FFPE) pairs of affected/unaffected tissue and its expression was assessed by real-time PCR. Two selected target genes, TRAF6 (tumour necrosis factor receptor-associated factor 6) and IRAK1 (Il-1 receptor-associated kinase 1/2), were also analysed. RESULTS: miR146a expression in FTC tissue was overall not downregulated in malignant versus unaffected tissue, but its expression was inversely correlated with clinicopathological features of FTCs at diagnosis. A decreased expression of miR146a became apparent in FTC thyroid tissue of widely compared to minimally invasive tumours. However, miR146a expression differences between contralateral unaffected tissue (extra-FTC) and FTC were not observed regardless of clinicopathological features. IRAK1, a known target for miR146a, was upregulated in FTC and the increase was mainly appreciable in Hurtle FTC variant. Unexpectedly, miR146a did not correlate with TRAF6 showing an inverse trend compared to IRAK1 although both genes regulate the activity of nuclear factor- kB (NF-kB). CONCLUSION: The results of this study indicate that downregulation of miR146a, inversely correlated with clinicopathological features of FTCs at diagnosis and suggest a possible involvement of miR146a in FTC development. IRAK1 over-expression in FTC may be related to tumour development/progression. In vitro experiments are needed to support this hypothesis.

2018 - Characteristics of a nationwide cohort of patients presenting with isolated hypogonadotropic hypogonadism (IHH) [Articolo su rivista]
Bonomi, Marco; Vezzoli, Valeria; Krausz, Csilla; Guizzardi, Fabiana; Vezzani, Silvia; Simoni, Manuela; Bassi, Ivan; Duminuco, Paolo; Di Iorgi, Natascia; Giavoli, Claudia; Pizzocaro, Alessandro; Russo, Gianni; Moro, Mirella; Fatti, Letizia; Ferlin, Alberto; Mazzanti, Laura; Zatelli, Maria Chiara; Cannavò, Salvo; Isidori, Andrea M.; Pincelli, Angela Ida; Prodam, Flavia; Mancini, Antonio; Limone, Paolo; Tanda, Maria Laura; Gaudino, Rossella; Salerno, Mariacarolina; Francesca, Pregnolato; Maghnie, Mohamad; Maggi, Mario; Persani, Luca; Aimaretti, G.; Altobell, M.; Ambrosio, M. R.; Andrioli, M.; Angelett, G.; Arecco, F.; Arnald, G.; Arosio, M.; Balsamo, A.; Baldassarr, M.; Bartalena, L.; Bazzon, N.; Beccari, L.; Beck-Peccoz, P.; Bellastella, G.; Bellizz, M.; Benedicent, F.; Bernasconi, S.; Bizzarri, C.; Bona, G.; Bonadonna, S.; Borrett, G.; Boschetti, M.; Brunani, A.; Brunelli, V.; Buz, F.; Cacciatore, C.; Cangiano, B.; Cappa, M.; Casalone, R.; Cassio, A.; Cavarzere, P.; Cherubini, V.; Ciampani, T.; Cicognan, D.; Cignarell, A.; Cisternin, M.; Colombo, P.; Corbetta, S.; Corciul, N.; Corona, G.; Cozzi, R.; Crivellaro, C.; Dalle Mule, I.; Danesi, L.; Eli, A. V. D.; Degli Uberti, E.; De Leo, S.; Della Valle, E.; De Marchi, M.; Di Iorgi, N.; Di Mambr, A.; Fabbri, A.; Foresta, C.; Forti, G.; Franceschi, A. R.; Garolla, A.; Ghezzi, M.; Giacomozzi, C.; Giusti, M.; Grosso, E.; Guabello, G.; Guarneri, M. P.; Grugni, G.; Isidori, A. M.; Lanfranco, F.; Lania, A.; Lanzi, R.; Larizza, L.; Lenzi, A.; Loche, S.; Loli, P.; Lombardi, V.; Maggi, M. C.; Mandrile, G.; Manieri, C.; Mantovani, G.; Marelli, S.; Marzullo, M.; Mencarelli, M. A.; Migone, N.; Motta, G.; Neri, G.; Padov, G.; Parenti, G.; Pasquino, B.; Pia, A.; Piantanida, E.; Pignatti, E.; Pilotta, A.; Pivett, B.; Pollazzon, M.; Pontecorvi, A.; Porcelli, P.; Pozza, G. B.; Pozzobon, G.; Radetti, G.; Razzore, P.; Rocchett, L.; Roncoron, R.; Rossi, G.; Sala, E.; Salvatoni, A.; Salvini, F.; Secc, A.; Segni, M.; Selice, R.; Sgaramella, P.; Sileo, F.; Sinisi, A. A.; Sirchia, F.; Spada, A.; Tresoldi, A.; Vigneri, R.; Weber, G.; Zucchini, S.
abstract

Objective: Isolated hypogonadotropic hypogonadism (IHH) is a rare disorder with pubertal delay, normal (normoosmic-IHH, nIHH) or defective sense of smell (Kallmann syndrome, KS). Other reproductive and nonreproductive anomalies might be present although information on their frequency are scanty, particularly according to the age of presentation. Design: Observational cohort study carried out between January 2008 and June 2016 within a national network of academic or general hospitals. Methods: We performed a detailed phenotyping of 503 IHH patients with: (1) manifestations of hypogonadism with low sex steroid hormone and low/normal gonadotropins; (2) absence of expansive hypothalamic/pituitary lesions or multiple pituitary hormone defects. Cohort was divided on IHH onset (PPO, pre-pubertal onset or AO, adult onset) and olfactory function: PPO-nIHH (n = 275), KS (n = 184), AO-nIHH (n = 36) and AO-doIHH (AO-IHH with defective olfaction, n = 8). Results: 90% of patients were classifed as PPO and 10% as AO. Typical midline and olfactory defects, bimanual synkinesis and familiarity for pubertal delay were also found among the AO-IHH. Mean age at diagnosis was signifcantly earlier and more frequently associated with congenital hypogonadism stigmata in patients with Kallmann's syndrome (KS). Synkinesis, renal and male genital tract anomalies were enriched in KS. Overweight/obesity are signifcantly associated with AO-IHH rather than PPO-IHH. Conclusions: Patients with KS are more prone to develop a severe and complex phenotype than nIHH. The presence of typical extra-gonadal defects and familiarity for PPO-IHH among the AO-IHH patients indicates a common predisposition with variable clinical expression. Overall, these fndings improve the understanding of IHH and may have a positive impact on the management of patients and their families.

2017 - Effects of chronic administration of the phosphodiesterase inhibitor vardenafil on serum levels of adrenal and testicular steroids in men with type 2 diabetes mellitus [Articolo su rivista]
Santi, Daniele; Granata, A. R.; Pignatti, Elisa; Trenti, T.; Roli, L.; Bozic, R.; Zaza, S.; Pacchioni, C.; Rochira, Vincenzo; Carani, Cesare; Simoni, Manuela
abstract

To investigate whether long-term, chronic treatment with the phosphodiesterase-5 inhibitor vardenafil affects adrenal and testicular steroidogenesis in diabetic men, using liquid chromatography-tandem mass spectrometry. A longitudinal, prospective, investigator-started, randomized, placebo-controlled, double-blind, clinical-trial was carried out, enrolling 54 male patients affected by type 2 diabetes mellitus diagnosed within the last 5 years. In total, 26 and 28 patients were followed for 1 year and assigned to the study and placebo group, respectively. Progesterone, 17-hydroxyprogesterone, androstenedione, testosterone, dehydroepiandrosterone, dehydroepiandrosterone sulfate, corticosterone, 11-deoxycortisol and cortisol, were evaluated using liquid chromatography-tandem mass spectrometry. No differences were seen in sex testicular steroids between study and control group. As for the adrenal gland, steroids were considered according to the zona in which they are produced. No significant differences were seen in steroid produced in zona fasciculata. For the zona reticularis, dehydroepiandrosterone significantly decreased during treatment only in the study group (p = 0.007), with higher levels at visit 2 and 8 than other visits. The dehydroepiandrosterone sulfate/dehydroepiandrosterone ratio significantly increased during treatment only in the verum group. Considering the adrenal zona glomerulosa, corticosterone significantly changed among visits both in both groups (p < 0.001), with higher levels at visit 2 (p = 0.028), 8 (p = 0.003), and 10 (p = 0.044), i.e., in coincidence with the complete clinical and instrumental examination performed only at these visits according to the study protocol. Chronically administered vardenafil reduces dehydroepiandrosterone levels and increases dehydroepiandrosterone sulfate/dehydroepiandrosterone ratio as possible consequences of modulation of steroidogenic enzymes by tissue changes in cyclic adenosine monophosphate and cyclic guanosine monophosphate availability. A possibly stress-related increase in corticosterone is suggested for the first time.

2017 - Heterogeneous hCG and hMG commercial preparations result in different intracellular signalling but induce a similar long-term progesterone response in vitro [Articolo su rivista]
Riccetti, Laura; Klett, Danièle; Ayoub, Mohammed Akli; Boulo, Thomas; Pignatti, Elisa; Tagliavini, Simonetta; Varani, Manuela; Trenti, Tommaso; Nicoli, Alessia; Capodanno, Francesco; La Sala, Giovanni Battista; Reiter, Eric; Simoni, Manuela; Casarini, Livio
abstract

STUDY QUESTION: Are four urinary hCG/menotropin (hMG) and one recombinant preparation characterized by different molecular features and do they mediate specific intracellular signaling and steroidogenesis?SUMMARY ANSWER: hCG and hMG preparations have heterogeneous compositions and mediate preparation-specific cell signaling and early steroidogenesis, although similar progesterone plateau levels are achieved in 24 h-treated human primary granulosa cells in vitro.WHAT IS KNOWN ALREADY: hCG is the pregnancy hormone marketed as a drug for ARTs to induce final oocyte maturation and ovulation, and to support FSH action. Several hCG formulations are commercially available, differing in source, purification methods and biochemical composition.STUDY DESIGN, SIZE, DURATION: Commercial hCG preparations for ART or research purposes were compared in vitro.PARTICIPANTS/MATERIALS, SETTING, METHODS: The different preparations were quantified by immunoassay with calibration against the hCG standard (Fifth IS; NIBSC 07/364). Immunoreactivity patterns, isoelectric points and oligosaccharide contents of hCGs were evaluated using reducing and non-reducing Western blotting, capillary isoelectric-focusing immunoassay and lectin-ELISA, respectively. Functional studies were performed in order to evaluate intracellular and total cAMP, progesterone production and beta-arrestin 2 recruitment by ELISA and BRET, in both human primary granulosa lutein cells (hGLC) and luteinizing hormone (LH)/hCG receptor (LHCGR)-transfected HEK293 cells, stimulated by increasing hormone concentrations. Statistical analysis was performed using two-way ANOVA and Bonferroni post-test or Mann-Whitney's U-test as appropriate.MAIN RESULTS AND THE ROLE OF CHANCE: Heterogeneous profiles were found among preparations, revealing specific molecular weight patterns (20-75 KDa range), isoelectric points (4.0-9.0 pI range) and lectin binding (P < 0.05; n = 7-10). These drug-specific compositions were linked to different potencies on cAMP production (EC50 1.0-400.0 ng/ml range) and beta-arrestin 2 recruitment (EC50 0.03-2.0 mu g/ml) in hGLC and transfected HEK293 cells (P < 0.05; n = 3-5). In hGLC, these differences were reflected by preparation-specific 8-h progesterone production although similar plateau levels of progesterone were acheived by 24-h treatment (P >= 0.05; n = 3).LARGE SCALE DATA: N/A.LIMITATIONS, REASONS FOR CAUTION: The biological activity of commercial hCG/hMG preparations is provided in International Units (IU) by in-vivo bioassay and calibration against an International Standard, although it is an unsuitable unit of measure for in-vitro studies. The re-calibration against recombinant hCG, quantified in grams, is based on the assumption that all of the isoforms and glycosylation variants have similar immunoreactivity.WIDER IMPLICATIONS OF THE FINDINGS: hCG/hMG preparation-specific cell responses in vitro may be proposed to ART patients affected by peculiar ovarian response, such as that caused by polycystic ovary syndrome. Otherwise, all the preparations available for ART may provide a similar clinical outcome in healthy women.

2016 - Effects of chronic administration of the phosphodiesterase inhibitor vardenafil on serum levels of adrenal and testicular steroids in men with type 2 diabetes mellitus [Abstract in Rivista]
Santi, Daniele; Granata, A. R. M.; Pignatti, Elisa; Trenti, T.; Roli, L.; Bozic, R.; Zaza, S.; Rochira, Vincenzo; Carani, Cesare; Magnani, Elisa; Simoni, Manuela
abstract

Background. Steroidogenesis is a complex enzymatic pro- cess in which cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) play an important role. Phosphodiesterase-5 inhibitors(PDE5i) increase cGMP, improving NO availability. Objective. To investigate whether long-term, chronic treat- ment with the PDE5i Vardenafil affects adrenal and testicular steroidogenesis in diabetic men, using liquid chromatography- mass spectrometry (LC-MS/MS). Design. A longitudinal, prospective, investigator-started, randomized, placebo-controlled, double-blind, clinical-trial was carried out. Setting and participants. 54 male patients affected by T2DM diagnosed within the last 5 years were enrolled. 26 and 28 patients were assigned to the verum and placebo-group, re- spectively. Interventions. The study consisted of an enrolment phase, a treatment phase (24 weeks) (Vardenafil/placebo 10 mg twice- daily), and a follow-up phase (24 weeks). Outcome measurements. Progesterone (P), 17-hydroxy- progesterone (17OHP), androstenedione (A), testosterone (T), dehydroepiandrosterone (DHEA), DHEA sulphate (DHEAS), corticosterone, 11-deoxycortisol and cortisol (C), were evalu- ated using LC-MS/MS. Results. No differences were seen in sex testicular steroids between study and control group. For the adrenal gland, steroids were considered according to the zona in which they are pro- duced. Considering steroids produced in the zona fasciculata, no significant differences were seen in 11-deoxycortisol and C among visits, both in the study and in the control group. For the zona reticularis, DHEA significantly decreased during treatment only in the study group (p=0.007). At post-hoc test DHEA showed higher levels at visit 2 and 8 than in other visits. The DHEAS/DHEAS ratio significantly increased during treatment only in the verum group. Considering the adrenal zona glomeru- losa, corticosterone significantly changed among visits both in the study and in the control group (p<0.001). At post-hoc test, in ПРОБЛЕМЫ ЭНДОКРИНОЛОГИИ, 5, 2016 both groups, corticosterone was significantly higher at visit 2 (p=0.028), 8 (p=0.003) and 10 (p=0.044), i.e. in coincidence with the complete clinical and instrumental examination per- formed only at these visits according to the study protocol. Conclusions. This is the first double-blind, placebo-con- trolled clinical-trial in which steroidogenesis is extensively in- vestigated by LC-MS/MS in T2DM men chronically treated with Vardenafil for 6 months, and followed-up for 6 months after therapy-withdrawal. Chronically administered Vardenafil reduces DHEA levels and increases DHEAS/DHEA ratio as possible consequences of modulation of steroidogenic enzymes by tissue changes in cGMP and/or cAMP availability. A possi- bly stress-related increase in corticosterone is suggested for the first time.

2016 - Six months of daily treatment with Vardenafil improves parameters of endothelial inflammation and of hypogonadism in male patients with type 2 diabetes and erectile dysfunction: a randomized, double-blind, prospective trial. [Articolo su rivista]
Santi, Daniele; Granata, Ar; Guidi, Alessandro; Pignatti, Elisa; Trenti, T; Roli, L; Bozic, R; Zaza, S; Pacchioni, C; Romano, S; Nofer, Jr; Rochira, Vincenzo; Carani, Cesare; Simoni, Manuela
abstract

Abstract OBJECTIVE: Type 2 diabetes mellitus (T2DM) is associated with endothelial dysfunction, characterized by a reduction of nitric oxide (NO)-mediated relaxation. Phosphodiesterase-5 inhibitors (PDE5i) improve NO levels. The aim of the study is to investigate whether long-term, chronic treatment with the PDE5i Vardenafil improves systemic endothelial function in diabetic men. DESIGN: Prospective, investigator-initiated, randomized, placebo-controlled, double-blind, clinical-trial. METHODS: 54 male patients affected by T2DM, diagnosed within the last 5 years, and erectile dysfunction were enrolled, regardless of testosterone (T) levels. 26 and 28 patients were assigned to verum and placebo-group, respectively. The study consisted of an enrolment phase, a treatment phase (24weeks) (Vardenafil/placebo 10mg twice-daily), and a follow-up phase (24weeks). Parameters evaluated: International Index of Erectile Function (IIEF)-15, flow mediated dilation (FMD), serum interleukin (IL)-6, endothelin (ET)-1, gonadotropins and T (measured by liquid-chromatography/tandem mass-spectrometry). RESULTS: IIEF-15 erectile function improved during treatment (p<0.001). At the end of the treatment both FMD (p=0.040) and IL-6 (p=0.019) significantly improved. FMD correlated with serum T levels (R2=0.299, p<0.001). T increased significantly under Vardenafil treatment and returned in the eugonadal range only in hypogonadal men (n=13), without changes in gonadotropins. Chronic Vardenafil treatment did not result in relevant side effects. CONCLUSIONS: This is the first double-blind, placebo-controlled clinical-trial designed to evaluate the effects of chronic treatment of Vardenafil on endothelial health-related parameters and sexual hormones in patients affected by a chronic disease. Chronically administered Vardenafil is effective and improves endothelial parameters in T2DM patient. Moreover, chronic Vardenafil therapy improves hypogonadism in diabetic, hypogonadal men.

2016 - Treatment with human, recombinant FSH improves sperm DNA fragmentation in idiopathic infertile men depending on the FSH receptor polymorphism p.N680S: A pharmacogenetic study [Articolo su rivista]
Simoni, Manuela; Santi, Daniele; Negri, Luciano; Hoffmann, Ivan; Muratori, Monica; Baldi, Elisabetta; Cambi, Marta; Marcou, Marios; Greither, Thomas; Baraldi, Enrica; Tagliavini, Simonetta; Carra, Daniela; Lombardo, Francesco; Gandini, Loredana; Pallotti, Francesco; Krausz, Csilla; Rastrelli, Giulia; Ferlin, Alberto; Menegazzo, Massimo; Pignatti, Elisa; Linari, Francesca; Marino, Marco; Benaglia, Renzo; Levi Setti, Paolo E.; Behre, Hermann M.
abstract

Study question: Does the spermDNAfragmentation index (DFI) improve depending on the FSH receptor (FSHR) genotype as assessed by the nonsynonymous polymorphisms rs6166 (p.N680S) after 3 months of recombinant FSH treatment in men with idiopathic infertility? summary answer: FSH treatment significantly improves sperm DFI only in idiopathic infertile men with the p.N680S homozygous N FSHR. what is known already: FSH, fundamental for spermatogenesis, is empirically used to treat male idiopathic infertility and several studies suggest that DFI could be a candidate predictor of response to FSH treatment, in terms of probability to conceive. Furthermore, it is known that the FSHR single nucleotide polymorphism (SNP) rs6166 (p.N680S) influences ovarian response in women and testicular volume in men. study design, size and duration: Amulticenter, longitudinal, prospective, open-label, two-arm clinical trial was performed. Subjects enrolled were idiopathic infertile men who received 150 IU recombinant human FSH s.c. every other day for 12 weeks and were followed-up for a further 12 weeks after FSH withdrawal. Patients were evaluated at baseline, at the end of treatment and at the end of follow-up. participants/materials, setting, methods: Eighty-nine men with idiopathic infertility carrier of the FSHR p.N680S homozygousNor S genotype, FSH ≤ 8 IU/l and DFI >15%,were enrolled. A total of 66 patients had DFI analysis completed on at least two visits. DFI was evaluated in one laboratory by TUNEL/PI (propidium iodide) assay coupled to flow cytometry, resolving two different fractions of sperm, namely the 'brighter' and 'dimmer' sperm DFI fractions. main results and the roleof chance: Thirty-eightmen(57.6%)were carriers of the p.N680S homozygousNand 28 (42.4%) of the homozygous S FSHR. Sperm concentration/number was highly heterogeneous and both groups included men ranging from severe oligozoospermia to normozoospermia. Total DFI was significantly lower at the end of the study in homozygous carriers of the p.N680SNversus p.N680S S allele (P = 0.008). Total DFI decreased significantly from baseline to the end of the study (P = 0.021) only in carriers of the p.N680S homozygous N polymorphism, and this decrease involved the sperm population containing vital sperm (i.e. brighter sperm) (P = 0.008). The dimmer sperm DFI fraction, including only nonvital sperm, was significantly larger in p.N680S S homozygous patients than in homozygous N men (P = 0.018). Total DFIwas inversely related to total sperm number (P = 0.020) and progressive sperm motility (P = 0.014).Whenpatients were further stratified according to sperm concentration (normoozospermic versus oligozoospermic) or -211G>T polymorphism in the FSHB gene (rs10835638) (homozygous Gversus others), the significant improvement of sperm DFI in FSHR p.N680S homozygousNmen was independent of sperm concentration and associated with the homozygous FSHB -211G>T homozygous G genotype. limitations, reasons for caution: The statistical power of the study is 86.9% with alpha error 0.05. This is the first pharmacogenetic study suggesting that FSH treatment induces a significant improvement of total DFI in men carriers of the p.N680S homozygousNFSHR; however, the results need to be confirmed in larger studies using a personalized FSH dosage and treatment duration. wider implications of the findings: The evaluation of sperm DFI as a surrogate marker of sperm quality, and of the FSHR SNP rs6166 (p.N680S), might be useful to predict the response to FSH treatment in men with idiopathic infertility. study funding/competing interest(s): The study was supported by an unrestricted grant to M.S. and H.M.B. from Merck Serono that provided the drug used in the study. MS received additional grants from Merck Serono and IBSA as well as honoraria from Merck Serono. The remaining authors declare that no conflicts of interest are present. trial registration number: EudraCT number 2010-020240-35.

2015 - Chronic, long-term administration of Vardenafil improves endothelial function and corrects hypogonadism in patients with type 2 diabetes mellitus. A longitudinal, prospective, randomized, placebo-controlled, double blind, clinical trial [Abstract in Rivista]
Santi, Daniele; Guidi, Alessandro; Granata, Antonio; Pignatti, Elisa; Bozic, Roberto; Zaza, Stefano; Roli, Laura; Trenti, Tommaso; Carani, Cesare; Simoni, Manuela
abstract

10.1530/endoabs.37.OC4.3

2015 - FSH treatment improves sperm DNA damage in men with idiopathic infertility carriers of the FSH receptor p.N680S homozygous N genotype: an interim analysis [Abstract in Atti di Convegno]
Simoni, M; Santi, D; Linari, F; Baldi, E; Cambi, M; Ferlin, A; Gandini, L; Garolla, A; Krausz, C; Levi Setti, Pe; Lombardo, F; Marino, M; Muratori, M; Negri, L; Pignatti, E; Rastrelli, G; and Behre, Hm
abstract

Study question: To assess whether in men with idiopathic infertility, the sperm DNA fragmentation (sDF) improves depending on the FSH receptor (FSHR) genotype as assessed by the non-synonymous polymorphisms (SNP) rs6166 (wild type or p.N680S). Summary answer: FSH treatment improves sDF in a subgroup of idiopathic infertile men, although 40% of these men do not show any significant improve- ment. The response of sDF, a surrogate marker of sperm quality, together with the evaluation of FSHR SNP p.N680S might be useful to predict the response to FSH treatment. What is known already: FSH is fundamental for spermatogenesis and is em- pirically used to treat male idiopathic infertility. Several studies suggest that sDF could be a candidate predictor of response to FSH treatment, in terms of probability to conceive. Furthermore, it is widely accepted that the FSHR SNP p.N680S influences ovarian response in women and testicular volume in men. Study design, size, duration: Multicenter, longitudinal, prospective, open-la- bel, two-arms clinical trial. Subjects enrolled were idiopathic infertile men and received 150 IU of recombinant FSH (Gonal f®) every other day for 12 weeks and were then followed-up for further 12 weeks after FSH-withdrawal. Patients were evaluated at baseline and at the end of the two phases. Participants/materials, setting, methods: Eighty-eight men with idiopathic male infertility carrier of the homozygous FSHR p.N680S N or S genotype, FSH < 8 IU/L and sDF > 15%, were enrolled. 66 patients completed the sDF analysis. sDF was centrally evaluated by TUNEL/PI assay coupled to flow cy- tometry, resolving two different sperm populations, namely: PIbrighter and PIdimmer. Main results and the role of chance: Thirty-seven men (56%) were carriers of the p.N680S homozygous-N and 29 (44%) of the homozygous-S genotype, respectively. Total sDF (PIbrighter + PIdimmer) was significantly lower at the end of the study in patients carriers of the p.N680S-N allele than patients carri- ers of p.N680S-S allele (p = 0.008). Only in patients carriers of the p.N680S-N allele, total sDF decreased significantly from baseline to the end of the study (p = 0.021) and this decrease was entirely sustained by the sperm population containing vital sperms (i.e., PIbrighter fraction) (p = 0.008). PIdimmer frac- tion, including only non-vital sperms, was significantly higher in patients car- riers of the p.N680S-S allele than in carriers of N allele (p = 0.018). Total sDF was inversely related to total sperm number (p = 0.020) and progressive sperm motility (p = 0.014). Limitations, reason for caution: The statistical power of the results obtained so far is 86.9%, with alpha-error 0.05. This is an interim-analysis. Wider implications of the findings: The study suggests that FSH treatment induces a significant improvement of total sDF in men carriers of the p.N680S homozygous N allele. This sDF decrease awaits confirmation, since the study will be completed by June 2015. Study funding/competing interest(s): Funding by commercial/corporate company(ies) – The study was supported by unrestricted grant by Merck Serono. Trial registration number: EudraCT number 2010-020240-35. Keywords: FSH treatment, male infertility, Sperm-DNA fragmentation

2015 - High-Resolution Melting is a sensitive, cost-effective, time-saving technique for BRAF V600E detection in thyroid FNAB washing fluid: a prospective cohort study [Articolo su rivista]
Marino, Marco; Monzani, Maria Laura; Brigante, Giulia; Cioni, Katia; Madeo, Bruno; Santi, Daniele; Maiorana, Antonino; Bettelli, Stefania Raffaella; Moriondo, Valeria; Pignatti, Elisa; Bonacini, Lara; Carani, Cesare; Rochira, Vincenzo; Simoni, Manuela
abstract

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2015 - Telomerase in differentiated thyroid cancer: promoter mutations, expression and localization [Articolo su rivista]
Muzza, Marina; Colombo, Carla; Rossi, Stefania; Tosi, Delfina; Cirello, Valentina; Perrino, Michela; De Leo, Simone; Magnani, Elisa; Pignatti, Elisa; Vigo, Beatrice; Simoni, Manuela; Bulfamante, Gaetano; Vicentini, Leonardo; Fugazzola, Laura
abstract

Telomerase-reverse-transcriptase (TERT) promoter mutations have been recently described in tumors. In the present large series, TERT mutations were found in 12% of papillary thyroid cancers (PTCs) and in 14% of follicular thyroid cancers (FTCs), and were found to significantly correlate with older age at diagnosis and poorer outcome. Interestingly, the prognostic value of TERT mutations resulted to be significantly stronger than that of BRAF(V600E). Moreover, the outcome was not different among tumors with isolated TERT mutation and those with coexistent mutations (TERT/BRAF in PTCs or TERT/RAS in FTCs). TERT rs2853669 polymorphism was found in 44.4% of tumors. At WB, TERT was significantly more expressed in tumors than in normal samples, being the highest levels of expression recorded in TERT mutated cases. At IHC, in tumors and in metastatic lymph-nodes TERT staining was significantly higher in the cytoplasm than in the nucleus, whereas in normal tissue the degree of staining did not differ in the two cellular compartments. In conclusion, TERT mutations were shown to strongly correlate with a poorer outcome in differentiated thyroid tumors, and neither BRAF nor RAS mutation were found to confer an additional effect in the disease persistence. TERT protein was found to be more expressed in neoplastic than in normal tissues, and to display a different cellular localization, suggesting that it could contribute to thyroid cancer progression by mechanisms taking place in the cytoplasm.

2014 - BRAF V600E mutation in washing liquid of thyroid fine-needle aspiration: a surprising tool in cytological benign nodules. [Abstract in Rivista]
Monzani, Maria Laura; Brigante, Giulia; Marino, Marco; L., Bonacini; Pignatti, Elisa; K., Cioni; Madeo, Bruno; Rochira, Vincenzo; Santi, Daniele; Maiorana, Antonino; Carani, Cesare; Simoni, Manuela
abstract

The good diagnostic value of a new method for BRAF V600E detection within the washing fluid of thyroid fine-needle aspiration biopsy has been demonstrated together with time and cost saving characteristics of this procedure.

2014 - Central hypogonadotropic hypogonadism: genetic complexity of a complex disease [Articolo su rivista]
Marino, Marco; Moriondo, Valeria; Vighi, Eleonora; Pignatti, Elisa; Simoni, Manuela
abstract

Central hypogonadotropic hypogonadism (CHH) is an emerging pathological condition frequently associated with overweight, metabolic syndrome, diabetes, and midline defects. The genetic mechanisms involve mutations in at least twenty-four genes regulating GnRH neuronal migration, secretion, and activity. So far, the mechanisms underlying CHH, both in prepubertal and in adulthood onset forms, remain unknown in most of the cases. Indeed, all detected gene variants may explain a small proportion of the affected patients (43%), indicating that other genes or epigenetic mechanisms are involved in the onset of CHH. The aim of this review is to summarize the current knowledge on genetic background of CHH, organizing the large amount of data present in the literature in a clear and concise manner, to produce a useful guide available for researchers and clinicians.

2014 - Espressione di miRNA-146a nel carcinoma follicolare della tiroide e correlazione con istotipo e stadiazione clinica [Abstract in Atti di Convegno]
Pignatti, Elisa; Vighi, Eleonora; Magnani, Elisa; Kara, Elda; Maiorana, Antonino; Rochira, Vincenzo; Carani, Cesare; Simoni, Manuela
abstract

This study investigates the relationship among miRNA-146a and clinical and histological correlates in follicular thyroid cancer

2014 - FSH treatment improves sperm DNA damage in men with idiopathic infertility carriers of the FSH receptor p.N680S homozygous N genotype: an interim analysis [Abstract in Atti di Convegno]
Santi, Daniele; Linari, Francesca; Baldi, E; Behre, Hm; Cambi, M; Ferlin, A; Gandini, L; Garolla, A; Krausz, C; Levi Setti, Pe; Lombardo, F; Marino, M; Muratori, M; Negri, L; Pignatti, E; Rastrelli, G; Simoni, Manuela
abstract

FSH treatment improves sperm DNA damage in men with idiopathic infertility carriers of the FSH receptor p.N680S homozygous N genotype: an interim analysis

2014 - Identificazione di BRAF V600E nel liquido di lavaggio dell’agoaspirato: un nuovo strumento nella diagnostica del nodulo tiroideo [Abstract in Atti di Convegno]
Monzani, Maria Laura; Brigante, Giulia; Marino, Marco; L., Bonacini; Pignatti, Elisa; K., Cioni; Madeo, Bruno; Rochira, Vincenzo; Santi, Daniele; Maiorana, Antonino; Carani, Cesare; Simoni, Manuela
abstract

This study investigates the diagnostic value of a new methodology for the detection of the BRAF mutation in samples of washing fluid of thyroid fine needle aspiration biopsies. The study demonstrates that this method is valid, and time and cost saving.

2014 - Pre-mir146a e FSHR sono marker di mosaicismo tiroideo nel carcinoma follicolare della tiroide [Abstract in Atti di Convegno]
Vighi, Eleonora; Pignatti, Elisa; Magnani, Elisa; Kara, Elda; Artuso, Lucia; Bernardis, Isabella; V., Cirello; Tagliafico, Enrico; Maiorana, Antonino; L., Fugazzola; Rochira, Vincenzo; Carani, Cesare; Simoni, Manuela
abstract

This study investigates the pre-mir146a within follicular thyroid cancer tissue and normal thyroid

2014 - Pre-miR146a expression in follicular carcinomas of the thyroid [Articolo su rivista]
Roncati, Luca; Pignatti, Elisa; Vighi, Eleonora; Magnani, Elisa; Kara, Elda; Rochira, Vincenzo; Carani, Cesare; Simoni, Manuela; Maiorana, Antonino
abstract

INTRODUCTION: Micro-RNA, a new class of small, non-coding RNAs, have been shown to be deregulated in several human carcinomas. In particular, SNP rs2910164 in pre-miR146a appears to be correlated with papillary thyroid carcinoma and may be involved in its genetic predisposition. Since data on follicular thyroid carcinomas (FTC) are lacking, we evaluated the involvement of SNP rs2910164 in FTC. METHODS: Thirty-nine cases of FTC and 20 follicular adenomas, defined according to WHO criteria, were selected. DNA and RNA were extracted from formalin-fixed paraffin-embedded blocks of both neoplastic and non-neoplastic areas. The DNA region of pre-miR146a, containing SNP rs2910164, was sequenced. Total RNA including miRNAs was used for stem-loop RT reactions, and applying a standard TaqMan PCR kit protocol for real-time PCR. Wilcoxon signed-rank test and Friedman test were used for statistical analyses. RESULTS: In 31% of FTC, the G allele was observed in neoplastic tissues, compared with the non-neoplastic areas (p < 0.05), whereas the CC phenotype was completely absent in tumours. Moreover, the expression of pre-miR146a was found to be significantly down-regulated in neoplastic tissues from FTC cases (p = 0.043), although no significant differences were seen in follicular thyroid adenomas. DISCUSSION: The expression profile of pre-miR146a can be correlated with FTC tumourigenesis. The G allele in SNP rs2910164 appears to be correlated with the transition from normal to neoplastic tissue. The GG and GC alleles appear to be associated with an increased risk for FTC, while the CC allele seems to play a protective role.

2014 - The TRHR Gene Is Associated with Hypothalamo-Pituitary Sensitivity to Levothyroxine [Articolo su rivista]
Brigante, Giulia; Spaggiari, Giorgia; Santi, Daniele; Cioni, Katia; Gnarini, Valentina; Diazzi, Chiara; Pignatti, Elisa; Casarini, Livio; Marino, Marco; Tüttelmann, Frank; Carani, Cesare; Simoni, Manuela
abstract

Thyroidectomized patients need variable doses of levothyroxine (LT4) to obtain target thyroid-stimulating hormone (TSH) levels. Individual feedback set-points have been hypothesized and the influence of several genes in the regulation of the pituitary-thyroid axis has been demonstrated.

2013 - Are pre-miR-146a and PTTG1 associated with papillary thyroid cancer? [Articolo su rivista]
Marino, Marco; Valentina, Cirello; Gnarini, Valentina; Carla, Colombo; Pignatti, Elisa; Casarini, Livio; Diazzi, Chiara; Rochira, Vincenzo; Katia, Cioni; Madeo, Bruno; Carani, Cesare; Simoni, Manuela; Laura, Fugazzola
abstract

Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy, with a steadily increasing incidence in the last few decades worldwide. The predisposition to developing this carcinoma by the heterozygous state of rs2910164 within the precursor of the miR-146a has been reported, but recently not confirmed. Interestingly, on the same chromosome, almost 50 kb separate the pre-miR-146a from the pituitary tumor-transforming gene 1 (PTTG1), a proto-oncogene involved in several tumors, including thyroid cancers. In this study, we analyzed, using a case–control design, the genetic association between PTC and the genomic region encompassing pre-miR-146a rs2910164 and PTTG1 rs1862391 and rs2910202. We enrolled 307 affected patients and 206 healthy controls. The possible presence of thyroid nodules in controls was excluded by ultrasonography. All the cases were submitted to single- nucleotide polymorphism (SNP) genotyping of pre-miR-146a and PTTG1, and risk association analyses were carried out. The genotypic and allelic frequencies of pre-miR-146a rs2910164 were not statistically different in the patients and controls, and this SNP was not in linkage disequilibrium with the investigated PTTG1 SNPs. Consistently, meta-analyses, the first including all the affected cases published to date, did not confirm the previously reported association of the heterozygous CG genotype with PTC. The PTTG1 SNPs exhibited the same allelic frequency in the patients and controls and were not associated with the disease. In conclusion, in a well-selected Italian population, neither pre-miR-146a rs2910164 nor PTTG1 rs1862391 and rs2910202 were found to be associated with the risk of developing PTC.

2013 - Association of pre-miR-146a rs2910164 GG genotype with papillary thyroid cancer: a new case control study on two adjacent genes on chromosome 5, pre-miR-146a and PTTG1 [Abstract in Rivista]
Marino, Marco; Valentina, Cirello; Gnarini, Valentina; Pignatti, Elisa; Casarini, Livio; Diazzi, Chiara; Rochira, Vincenzo; Katia, Cioni; Madeo, Bruno; Simoni, Manuela; Laura, Fugazzola
abstract

Role of the pre-miR-146a and PTTG1 on papilary thyroid cancer

2013 - Complete aromatase deficiency in four adult men: detection of a novel mutation and two known mutations in the CYP19A1 gene [Abstract in Rivista]
Pignatti, Elisa; K. M., Ajlouni; N., Khawaja; K., Unluhizarci; E., Kartal; Carani, Cesare; Simoni, Manuela; Marino, Marco; Vighi, Eleonora; Rochira, Vincenzo
abstract

The abstracts descibes four new cases of patients with aromatase deficiency. Both the clinical features and the results of the molecular studies are reported.

2013 - Complete aromatase deficiency in four adult men: detection of a novel mutation and two known mutations in the CYP19A1 gene [Abstract in Rivista]
Pignatti, Elisa; Kursad, Unluhizarci; Ermine, Kartal; Kamel, Ajlouni; Nahla, Khawaja; Carani, Cesare; Marino, Marco; Simoni, Manuela; Vighi, Eleonora; Rochira, Vincenzo
abstract

The abstract deals with the clinical and genetic description of 4 new cases of aromatase deficiency.

2013 - Complete aromatase deficiency in four adult men: detection of a novel mutation and two known mutations in the CYP19A1 gene [Abstract in Rivista]
Pignatti, Elisa; K., Ajlouni; N., Khawaja; K., Unluhizarci; E., Kartal; Carani, Cesare; Marino, Marco; Simoni, Manuela; Vighi, Eleonora; Rochira, Vincenzo
abstract

The study describes 4 new cases of the rare disease aromatase deficiency

2013 - Mutations and somatic changes in the genotype of rs2910164 in pre-miR146a are frequent in follicular thyroid carcinoma [Abstract in Rivista]
Vighi, Eleonora; Pignatti, Elisa; Roncati, Luca; Rochira, Vincenzo; Kara, Elda; Madeo, Bruno; E., Magnani; Maiorana, Antonino; Carani, Cesare; Simoni, Manuela
abstract

Mutations and somatic changes in the genotype of rs2910164 in pre-miR146a are frequent in follicular thyroid carcinoma and may be associated with its biological behavior

2013 - New insights on follicular thyroid carcinoma: the role of pre-mir-146a [Abstract in Rivista]
Pignatti, Elisa; Vighi, Eleonora; Roncati, Luca; E., Magnani; Kara, Elda; Madeo, Bruno; Rochira, Vincenzo; Maiorana, Antonino; Carani, Cesare; Simoni, Manuela
abstract

Role of pre-mir-146a on follicular thyroid cancer

2013 - Papillary thyroid cancer: a new case-control study involving pre-mir-146a and PTTG1 genes [Abstract in Rivista]
Marino, Marco; V., Cirello; Gnarini, Valentina; Pignatti, Elisa; Casarini, Livio; Diazzi, Chiara; Rochira, Vincenzo; K., Cioni; Madeo, Bruno; Simoni, Manuela; L., Fugazzola
abstract

Studio genetico dei carcinomi papillari della tiroide

2013 - Pre-miR146a expression profiling of follicular thyroid carcinoma [Abstract in Rivista]
Pignatti, Elisa; Vighi, Eleonora; Roncati, Luca; Kara, Elda; Eleonora, Porcheddu; Elisa, Magnani; Rochira, Vincenzo; Maiorana, Antonino; Carani, Cesare; Simoni, Manuela
abstract

Pre-miR146a may be a marker for predicitng the phenotype of thyroid follicular carcinomas

2013 - Prevalence of olfactory and other developmental anomalies in patients with central hypogonadotropic hypogonadism [Articolo su rivista]
DELLA VALLE, Elisa; Vezzani, Silvia; Rochira, Vincenzo; Antonio R. M., Granata; Madeo, Bruno; Genovese, Elisabetta; Pignatti, Elisa; Marino, Marco; Carani, Cesare; Simoni, Manuela
abstract

Introduction: Hypogonadotropic hypogonadism (HH) is a heterogeneous disease caused by mutations in several genes. Based on the presence of hyposmia/anosmia it is distin- guished into Kallmann syndrome (KS) and isolated HH. The prevalence of other develop- mental anomalies is not well established. Methods: We studied 36 patients with HH (31 males, 5 females, mean age 41.5), 9 with familial and 27 with sporadic HH (33 congenital, 3 adult-onset), by physical examination, smell test (BSIT Sensonics), audiometry, renal ultrasound, and magnetic resonance imag- ing of the olfactory structures. Results: Based on the smell test, patients were classified as normosmic (n = 21, 58.3%) and hypo/anosmic (n = 15, 41.6%). Hypoplasia/agenesis of olfactory bulbs was found in 40% of patients (10/25; 75% hypo/anosmic, 7.6% normosmic, p&lt;0.01, Fisher’s test). Remarkably, olfactory structures were normal in two anosmic patients, while one nor- mosmic patient presented a unilateral hypoplastic bulb. Fourteen of 33 patients (42.4%) presented neurosensorial hearing loss of various degrees (28.5% hypo/anosmic, 52.6% normosmic, p=NS). Renal ultrasound revealed 27.7% of cases with renal anomalies (26.6% hypo/anosmic, 28.5% normosmic, p = NS). At least one midline defect was found in 50% of the patients (53.3% hypo/anosmic, 47.6% normosmic, p = NS), including abnor- mal palate, dental anomalies, pectus excavatum, bimanual synkinesis, iris coloboma, and absent nasal cartilage. Anamnestically 4/31 patients reported cryptorchidism (25% hypo/anosmic, 5.2% normosmic, p = NS). Conclusion: Hypo/anosmia is significantly related to anatomical anomalies of the olfac- tory bulbs/tracts but the prevalence of other developmental anomalies, especially midline defects and neurosensorial hearing loss, is high both in HH and KS and independent of the presence of anosmia/hyposmia. From the clinical standpoint KS and normosmic HH should be considered as the same complex, developmental disease.

2013 - SNP RS2910164 in pre-mir146a undergoes somatic changes in follicular thyroid carcinoma [Abstract in Rivista]
Vighi, Eleonora; Pignatti, Elisa; Roncati, Luca; Rochira, Vincenzo; Kara, Elda; Madeo, Bruno; E., Magnani; Maiorana, Antonino; Carani, Cesare; Simoni, Manuela
abstract

Role of SNP in follicular thyroid cancer

2012 - Aromatase expression in human peripheral blood leukocytes (PBLs) and in various tissues in primates: studies in elderly humans and cynomolgus monkeys [Articolo su rivista]
Pignatti, Elisa; Casarini, Livio; S., Scaltriti; J., Wistuba; S., Schlatt; A., Rossi; A., Lachhab; E., Taliani; Carani, Cesare; Simoni, Manuela
abstract

Background Previous analysis of aromatase gene and protein expression in PBLs, studied in children and adults, were extended to elderly subjects. In addition we assessed whether aromatase expression in PBLs could be used as a parameter of aromatase expression in other tissues, using the cynomolgus monkey as model. Methods Real-time analysis of aromatase gene expression and protein evaluation by Western blot were performed in PBLs of human elderly subjects and in various tissues from cynomolgus monkeys. Results No gender-related difference in CYP19A1 mRNA and protein expression in PBLs from human elderly women and men was found. In elderly male cynomolgus monkeys CYP19A1 mRNA and protein were expressed in all cells and tissues analysed, with the lowest levels in PBLs but no clear-cut correlation with other tissues. Conclusions Aromatase expression in PBLs in elderly human subjects is not gender-related and cannot be a surrogate of aromatase expression for other tissues.

2012 - Effect of sphingosine 1-phosphate (S1P) receptor agonists FTY720 and CYM5442 on atherosclerosis development in LDL receptor deficient (LDL-R(-/-) mice [Articolo su rivista]
F., Potì; S., Costa; V., Bergonzini; M., Galletti; Pignatti, Elisa; C., Weber; Simoni, Manuela; J. R., Nofer
abstract

Objectives: Sphingosine 1-phosphate (S1P) – a lysosphingolipid present in HDL – exerts atheroprotective ef- fects in vitro, while FTY720, a non-selective S1P mimetic inhibits atherosclerosis in LDL receptor-deficient (LDL-R−/−) mice under conditions of severe hypercholesterolemia. We here examined the effect of FTY720 and a selective S1P receptor type 1 agonist CYM5442 on atherosclerosis in moderately hypercholesterolemic LDL-R−/− mice.Methods and results: LDL-R−/− mice fed Western diet (0.25% cholesterol) were given FTY720 (0.4 mg/kg/day) or CYM5442 (2.0 mg/kg/day) for 18 weeks. FTY720 but not CYM5422 persistently lowered blood lympho- cytes, depleted CD4+ and CD8+ T cells in spleen and lymph nodes, and reduced splenocyte IL-2 secretion. However, both compounds reduced the activity of splenic and peritoneal macrophages as inferred from the down-regulated CD68 and MHC-II expression in CD11b+ cells and the reduced IL-6 secretion in response to LPS, respectively. CYM5442 and FTY720 reduced weight gain, white adipose tissue depots and fasting glu- cose suggesting improvement of metabolic control, but failed to influence atherosclerosis in LDL-R−/− mice. Conclusion: Despite down-regulating macrophage function and – in case of FTY720 – altering lymphocyte dis- tribution CYM5442 and FTY720 fail to affect atherosclerosis in moderately hypercholesterolemic LDL-R−/− mice. We hypothesize that S1P mimetics exert atheroprotective effects only under conditions of increased cholesterol burden exacerbating vascular inflammation.

2012 - LH and hCG Action on the Same Receptor Results in Quantitatively and Qualitatively Different Intracellular Signalling [Articolo su rivista]
Casarini, Livio; Lispi, M.; Longobardi, S.; Milosa, F.; LA MARCA, Antonio; Tagliasacchi, Daniela; Pignatti, Elisa; Simoni, Manuela
abstract

Human luteinizing hormone (hLH) and chorionic gonadotropin (hCG) act on the same receptor (LHCGR) but it is not known whether they elicit the same cellular and molecular response. This study compares for the first time the activation of cell-signalling pathways and gene expression in response to hLH and hCG. Using recombinant hLH and recombinant hCG we evaluated the kinetics of cAMP production in COS-7 and hGL5 cells permanently expressing LHCGR (COS-7/LHCGR, hGL5/LHCGR), as well as cAMP, ERK1/2, AKT activation and progesterone production in primary human granulosa cells (hGLC). The expression of selected target genes was measured in the presence or absence of ERK- or AKT-pathways inhibitors. In COS-7/LHCGR cells, hCG is 5-fold more potent than hLH (cAMP ED50: 107.1±14.3 pM and 530.0±51.2 pM, respectively). hLH maximal effect was significantly faster (10 minutes by hLH; 1 hour by hCG). In hGLC continuous exposure to equipotent doses of gonadotropins up to 36 hours revealed that intracellular cAMP production is oscillating and significantly higher by hCG versus hLH. Conversely, phospho-ERK1/2 and -AKT activation was more potent and sustained by hLH versus hCG. ERK1/2 and AKT inhibition removed the inhibitory effect on NRG1 (neuregulin) expression by hLH but not by hCG; ERK1/2 inhibition significantly increased hLH- but not hCG-stimulated CYP19A1 (aromatase) expression. We conclude that: i) hCG is more potent on cAMP production, while hLH is more potent on ERK and AKT activation; ii) hGLC respond to equipotent, constant hLH or hCG stimulation with a fluctuating cAMP production and progressive progesterone secretion; and iii) the expression of hLH and hCG target genes partly involves the activation of different pathways depending on the ligand. Therefore, the LHCGR is able to differentiate the activity of hLH and hCG.

2012 - New understandings of the genetic basis of isolated idiopathic central hypogonadism [Articolo su rivista]
M., Bonomi; D. V., Libri; F., Guizzardi; E., Guarducci; E., Maiolo; Pignatti, Elisa; R., Asci; L., Persani; G., Aimaretti; M., Altobelli; G., Arnaldi; M., Baldi; L., Bartalena; L., Beccaria; P., Beck Peccoz; G., Bellastella; G., Borretta; F., Buzi; S., Cannavò; M., Cappa; A., Cariboni; T., Ciampani; A., Cicognani; M., Cisternino; S., Corbetta; N., Corciulo; R., Cozzi; A. V., D'Elia; E. D., Uberti; M., De Marchi; G., Forti; N., di Iorgi; A., Fabbri; A., Ferlin; R., Gaudino; E., Grosso; C., Krausz; F., Lanfranco; D., Larizza; P., Limone; M., Maggi; R., Maggi; M., Maghnie; A., Mancini; G., Mandrile; Marino, Marco; M. A., Mencarelli; N., Migone; G., Neri; L., Perroni; E., Pignatti; A., Pilotta; A. I., Pincelli; A., Pizzocaro; A., Pontecorvi; G., Radetti; P., Razzore; G., Russo; F., Salvini; A., Secco; M., Segni; Simoni, Manuela; A., Sinisi; R., Vigneri; G., Weber
abstract

Idiopathic hypogonadotropic hypogonadism is a rare disease that is characterized by delayed/absent puberty and/or infertility due to an insufficient stimulation of an otherwise normal pituitary-gonadal axis by gonadotrophin-releasing hormone (GnRH) action. Because reduced or normal luteinizing hormone (LH)/follicle-stimulating hormone (FSH) levels may be observed in the affected patients, the term idiopathic central hypogonadism (ICH) appears to be more appropriate. This disease should be distinguished from central hypogonadism that is combined with other pituitary deficiencies. Isolated ICH has a complex pathogenesis and is fivefold more prevalent in males. ICH frequently appears in a sporadic form, but several familial cases have also been reported. This finding, in conjunction with the description of numerous pathogenetic gene variants and the generation of several knockout models, supports the existence of a strong genetic component. ICH may be associated with several morphogenetic abnormalities, which include osmic defects that, with ICH, constitute the cardinal manifestations of Kallmann syndrome (KS). KS accounts for approximately 40% of the total ICH cases and has been generally considered to be a distinct subgroup. However, the description of several pedigrees, which include relatives who are affected either with isolated osmic defects, KS, or normo-osmic ICH (nICH), justifies the emerging idea that ICH is a complex genetic disease that is characterized by variable expressivity and penetrance. In this context, either multiple gene variants or environmental factors and epigenetic modifications may contribute to the variable disease manifestations. We review the genetic mechanisms that are presently known to be involved in ICH pathogenesis and provide a clinical overview of the 227 cases that have been collected by the collaborating centres of the Italian ICH Network.

2012 - The cellular fate of CYP19A1 (aromatase) protein [Abstract in Rivista]
Pignatti, Elisa; Ferioli, Silvia; Carani, Cesare; Rochira, Vincenzo; Francesco, Potì; Simoni, Manuela
abstract

Le variazioni nell'attività enzimatica e nella degradazione dell'enzima indotte dalle mutazioni finora descritte del gene CYP19A1 sono state studiate in vitro mediante studi di transfezione e confrontate con l'enzima wild type.

2012 - The TRHR gene is associated to hypothalamo-pituitary sensitivity to levothyroxine in thyroidectomized patients [Abstract in Atti di Convegno]
Brigante, Giulia; Spaggiari, Giorgia; Cioni, K; Gnarini, Valentina; Pignatti, Elisa; Casarini, Livio; Marino, Marco; Tüttelmann, F; Carani, Cesare; Simoni, Manuela
abstract

Background: Patients thyroidectomized for thyroid cancer need variable doses of levothyroxine (LT4) to obtain TSH suppression. A predetermined thyroid function set-point for each individual has been hypothesized, suggesting a genetic influence in the regulation of pituitary-thyroid axis. We hypothesized of the TRHR gene could be associated with a different hypothalamo-pituitary sensitivity to the negative feedback of the thyroid hormones. Methods: We performed a case–control association study, enrolling 107 thyroidectomized patients, in follow-up for differentiated thyroid cancer, and 99 volunteer controls. Patients were evaluated first when TSH levels were suppressed (<0.1 mIU/l), by the lowest effective LT4 dose, and then when TSH was subsuppressed (0.1<TSH<0.5 mIU/l). We selected two SNPs of TRHR gene, rs3134105 and rs3110040, identified as informative markers, using the online database ‘HapMap’. We performed a frequency analysis of the mapped SNPs, followed by a linkage analysis using the HaploView software. Genotyping was performed using the High Resolution Melting technology. Results: The selected SNPs were in linkage disequilibrium. A significant difference between the three possible genotypes for rs3134105 was found for fT4/TSH ratio (P=0.03). Moreover, despite similar serum concentrations of fT3 and fT4 obtained by similar levothyroxine doses, carriers of at least one A allele of rs3134105 had significantly lower serum TSH levels (P=0.04) as well as higher fT3/TSH (P=0.05) and fT4/TSH ratios (P=0.02). Conclusions: We demonstrated an association between TSH and discrete alleles of the TRHR gene identified by the markers SNPs rs3134105 and rs3110040 in totally thyroidectomized patients with diagnosis of thyroid cancer under subsuppressive LT4 therapy. The TRHR gene is a determinant of hypothalamo-pituitary sensitivity to levothyroxine in such patients.

2011 - Effects of polymorphisms in gonadotropin and gonadotropin receptor genes on reproductive function. [Articolo su rivista]
Casarini, Livio; Pignatti, Elisa; Simoni, Manuela
abstract

Gonadotropins, the action of which is mediated at the level of their gonadal receptors, play a key role in sexual development, reproductive functions and in metabolism. The involvement of the gonadotropins and their receptor genotypes on reproductive function are widely studied. A large number of gonadotropins and their receptors gene polymorphisms are known, but the only one considerable as a clear, absolute genetic marker of reproductive features or disfunctions is the FSHR Asn680Ser polymorphism, since it modulates ovarian response to FSH. The aim of these studies would to be the prediction of the genetic causes of sex-related diseases to enable a customized clinical setting based on individual response of patients undergoing gonadotropin stimulation. In this review we discuss the latest information about the effects of polymorphisms of the gonadotropins and their receptor genes on reproductive functions of both male and female, and discuss their patho-physiological implications.

2011 - Inactivating mutations of CYP19A1 (aromatase) gene reduce the protein stability in vitro [Abstract in Rivista]
Pignatti, Elisa; Ferioli, Silvia; Simoni, Manuela; Carani, Cesare; Rochira, Vincenzo
abstract

Role of inactivating mutation of the aromatase gene on the protein structure, function and degradation

2011 - Two hormone for one receptors: dissecting out LH and hCG activity with an in vitro approach [Abstract in Atti di Convegno]
Casarini, Livio; LA MARCA, Antonio; Pignatti, Elisa; Simoni, Manuela
abstract

Introduction: LH and hCG act on the same receptor (LHCGR), have different half-lives and in vivo biopotency. It is not known whether they elicit the same cellular and molecular response. The aim of this study was to compare the kinetics of cAMP response to recombinant LH and hCG. Design: In COS-7 cells permanently expressing the human LHCGR (COS-7/LHCGR) we evaluated LH and hCG dose-response curves, by measuring total cAMP after 3 h of incubation. We then evaluated the time-course of intracellular cAMP production in the presence of ED50 doses of LH and hCG over 3 h. Finally we evaluated the long-term response to LH and hCG by exposing human primary granulosa lutein cells (hGLC) to ED50 doses over 12 h. All incubations were performed in the presence of IBMX. Results: In COS-7/LHCGR cells, we observed significantly different ED50 for LH (475.75±137.33 pM, mean±S.D.) and hCG (101.75±44.63 pM) (Mann–Whitney’s U-test, P=0.029; n=4). Maximal LH stimulation of intracellular cAMP, about 50 fold over control, reached a plateau in 10 min, while maximal hCG stimulation at similar levels was attained only after 1 h (Anova; P<0.05; n=3). In hGLC continuous exposure to LH and hCG resulted in a repetitive, pulsatile increase of intracellular cAMP with peaks every 3–4 h and significantly higher levels of stimulation in the presence of hCG vs LH (Anova; P<0.05; n=3). Conclusions: Equimolar concentrations of human recombinant LH and hCG result in significantly higher in vitro biopotency of hCG (about 5-fold). Equipotent concentration (ED50) of LH and hCG stimulate a faster response to LH within the first 3 h, but a quantitatively higher response to hCG over 12 h. hGLC respond to constant LH/hCG stimulation in a pulsatile fashion, suggesting a novel control of gonadotropins action at the receptor level.

2009 - Aromatase expression in peripheral blood leukocytes from adult and elderly female and male subjects [Abstract in Rivista]
Pignatti, Elisa; Rossi, Aldo; Scaltriti, Sara; Taliani, Erica; Rochira, Vincenzo; Simoni, Manuela; Carani, Cesare
abstract

Study of the expression of aromatase enzyme in peripheral blood leukocytes collected from adult and elderly female and male subjects. This suds highlights the importance of individual differences in aromatase expression as well as the differences related to different age.

2008 - A novel compound heterozygous mutation of the aromatase gene in an adult man: new insights into the role of estrogen on gonadal development. [Abstract in Atti di Convegno]
Fabio, Lanfranco; Rochira, Vincenzo; Zirilli, Lucia; Matteo, Baldi; Pignatti, Elisa; Gianluca, Aimaretti; Carani, Cesare
abstract

Description of a new case of human aromatase deficiency in a man of 26 years of age and present the results of five year follow-up during trandermal estradiol (tE2) substitution, focusing on bone growth and mineralization

2008 - A novel compuond heterozigous mutation of the aromatase gene in adult man: new insights into the role of estrogen on gonadal development [Abstract in Rivista]
Fabio, Lanfranco; Zirilli, Lucia; Matteo, Baldi; Luberto, Alessandra; Pignatti, Elisa; Magnani, Francesca; Gianluca, Aimaretti; Carani, Cesare; Rochira, Vincenzo
abstract

Description of a new case of human aromatase deficiency in a man of 26 years of age and present the results of five year follow-up during trandermal estradiol (tE2) substitution, focusing on bone growth and mineralization

2008 - A novel mutation in the human aromatase gene: insights on the relationship among serum estradiol, longitudinal growth and bone mineral density in an adult man under estrogen replacement treatment. [Articolo su rivista]
Fabio, Lanfranco; Zirilli, Lucia; Matteo, Baldi; Pignatti, Elisa; Ginevra, Corneli; Ezio, Ghigo; Gianluca, Aimaretti; Carani, Cesare; Rochira, Vincenzo
abstract

Objective: Here we report on a new case of human aromatase de!ciency in a man of 26 years of age andpresent the results of !ve year follow-up during trandermal estradiol (tE2) substitution, focusing on bonegrowth and mineralization. The lack of patient's compliance to tE2 treatment, resulting in low but detectableserum estradiol levels, provides helpful information about the physiological estradiol needed in serum toguarantee a complete bone maturation and mineralization.Design: Clinical case report study.Methods: Genetic, biochemical and hormonal evaluations and the study of bone health were performedbefore and during estrogen treatment.Results: Eunuchoid body proportions, unfused epiphyses, tall stature, osteopenia, increase fasting insulin,mild astenozoospermia and a history of right cryptorchidism were present. Baseline serum FSH was slightlyabove the normal range and estradiol was undetectable. Genetic analysis revealed a pattern of compoundheterozygosity due to 23 bp deletion in exon IV and a point mutation in the !rst nucleotide of intron IX of theCYP19A1 gene, respectively. The closure of epiphyseal cartilage, the normalization of bone BMD and boneturnover markers, and the improvement of insulin levels were reached during tE2 only when serum estradiolraised above 73 pmol/L. Sperm parameters and overweight did not improve with substitutive therapy.Conclusions: This new case of aromatase de!ciency underlines the role of estrogen on skeletal maturation,BMD, metabolic abnormalities and gonadal axis. It provides evidence on the need not only of a continuousestrogen replacement, but also of ensuring adequate estradiol levels in serum in order to ensure a completebone maturation and mineralization and to prevent the worsening of body skeletal proportions. Thecomprehension of this physiological aspect has relevant clinical signi!cance especially for the developmentof new therapeutic strategies useful to treat growth disorders by targeting serum estradiol in men.

2008 - 21-hydroxylase deficiency and klinefelter syndrome in an adult man: striking a balance between androgen excess and insufficiency. [Articolo su rivista]
Balestrieri, Antonio; Zirilli, Lucia; Madeo, Bruno; Pignatti, Elisa; Rossi, Giulio; Carani, Cesare; Rochira, Vincenzo
abstract

Objective We describe the rare case of an adult man with normal virilization affected by both Klinefelter Syndrome (KS) and Congenital Adrenal Hyperplasia (CAH) due to 21-hydroxylase deficiency, consulting for painful gynecomastia. A complete clinical workup included endocrinological, genetic, sexological evaluation and testis histology. Genetic analyses included karyotype, CYP21 sequencing and the CAG and GGC repeat polymorphism in the androgen receptor gene. Findings KS was diagnosed by karyotype analysis (47,XXY), the testis biopsy revealed Leydig cell hyperplasia. The CAH was diagnosed by the direct detection of a I2 homozygous mutation in the CYP21 gene. The hormonal analysis revealed a mild hypergonadotropic hypogonadism, serum levels of androstenedione and ACTH above the normal range and a slightly reduced cortisol response with exaggerated 17-OH progesterone increase to ACTH stimulation. Cortisone acetate treatment disclosed a clinically relevant pre-existent hypogonadism in the relatively short time of 6 months, thus suggesting that the reduction in adrenal steroids impaired the balance in the androgen status previously created by the two syndromes. Only the combined therapy with cortisone acetate and testosterone restored a normal androgenization and a male sexual behavior. Conclusions The simultaneous occurrence of KS and CAH is extremely rare. The clinical phenotype of our patient was characterized by mild symptoms of the two syndromes, probably because the high levels of adrenal androgens due to CAH counterbalanced the partial androgen deficiency due to KS.

2007 - A novel compound heterozygous mutation of the aromatase gene in an adult man: a reinforced evidence on the relationships among congenital estrogen deficiency, adiposity and the metabolic syndrome [Abstract in Rivista]
Luberto, Alessandra; Rochira, Vincenzo; Zirilli, Lucia; Pignatti, Elisa; Evan R., Simpson; Laura E., Maffei; Carani, Cesare
abstract

The fourth case of an adult man affected by aromatase deficiency resulting from a novel homozygous inactivating mutation of the CYP19 (P450arom) gene.The patient presented a complex dysmetabolic syndrome that improved after estrogen replacement treatment

2007 - A novel compound heterozygous mutation of the aromatase gene in an adult man: new insight into the role of estrogen on gonadal development [Abstract in Rivista]
Luberto, Alessandra; Fabio, Lanfranco; Matteo, Baldi; Gianluca, Aimaretti; Rochira, Vincenzo; Pignatti, Elisa; Magnani, Francesca; Ezio, Ghigo; Carani, Cesare
abstract

Description of a new case of human aromatase deficiency in a man of 26 years of age and present the results of five year follow-up during trandermal estradiol (tE2) substitution, focusing on goandal development

2007 - A novel compound heterozygous mutation of the aromatase gene in an adult man: reinforced evidence on the relationship between congenital oestrogen deficiency, adiposity and the metabolic syndrome. [Articolo su rivista]
L., Maffei; Rochira, Vincenzo; Zirilli, Lucia; P., Antunez; C., Aranda; B., Fabre; M. L., Simone; Pignatti, Elisa; E. R., Simpson; S., Houssami; C. D., Clyne; Carani, Cesare
abstract

Abstract:Summary Background  Descriptions of new cases of human aromatase deficiency are useful for a better understanding of male oestrogen pathophysiology, as some aspects remain controversial. Objective  To present a new case of an adult man affected by aromatase deficiency, along with a description of clinical phenotype, and hormonal and genetic analysis. Design  Case report study. Patient  A 25-year-old man with continuing linear growth, eunuchoid body habitus and diffuse bone pain. Measurements  Amplification and sequencing of all coding exons with their flanking intronic sequences of the CYP19A1 gene. Aromatase expression of the mutant human cDNAs was compared with wild type. Serum LH, FSH, testosterone, oestradiol, insulin, glucose, glycosylated haemoglobin (HbA1c), serum lipids and liver enzymes were measured. Histological analysis of liver and testis biopsies was performed. Results  Two novel heterozygous compound inactivating mutations of the CYP19A1 gene were disclosed. The first mutation is at bp380 (T→G) in exon IV and the second one at bp 1124 (G→A) in exon IX. LH and testosterone were normal, FSH was slightly elevated, and serum oestradiol undetectable. The subject showed a metabolic syndrome characterized by abdominal obesity, hyperinsulinaemia, acanthosis nigricans and nonalcoholic fatty liver disease. Conclusions  These novel mutations improve our knowledge on genetics of the CYP19A1 gene. This new case of aromatase deficiency sheds new light on the heterogeneity of mutations in the CYP19A1 gene causing loss of function of the aromatase enzyme. The evidence of metabolic syndrome and of obesity associated with congenital oestrogen deprivation emphasizes the role of oestrogens in fat accumulation and distribution in men, a role that has long been partially overlooked in these patients.

2007 - Preliminary Results of Thyroid Disease Screening Program "Ambulatorio Mobile" of 6153 Thyroid Ultrasounds Performed on the Population of a Geographical Area in Belarus Contaminated by Chernobyl Accident. [Abstract in Atti di Convegno]
Zirilli, Lucia; Rochira, Vincenzo; Andrei, Alekseev; Natalia, Antanovich; Madeo, Bruno; Sanguanini, Alessia; Scaltriti, Sara; Romano, Stefania; Pignatti, Elisa; Massimotosti, Balducci; Carani, Cesare
abstract

The abstract deals with the effects of Chernobyl release of 131I on thyroid volume in subjects exposed to the fall-out. In particular no difference in thyroid volume was found as a consequence of 131I exposure.

2007 - The osteoporotic male: overlooked and undermanaged? [Articolo su rivista]
Madeo, Bruno; Zirilli, Lucia; Caffagni, Giovanni; Diazzi, Chiara; Sanguanini, Alessia; Pignatti, Elisa; Carani, Cesare; Rochira, Vincenzo
abstract

Age-related bone loss in men is a poorly understood phenomenon, although increasing data on the pathophysiology of bone in men is becoming available. Most of what we know on bone pathophysiology derives from studies on women. The well-known association between menopause and osteoporosis is far from been disproven. However, male osteoporosis is a relatively new phenomenon. Its novelty is in part compensated for by the number of studies on female osteoporosis and bone pathophysiology. On the other hand, the deeper understanding of female osteoporosis could lead to an underestimation of this condition in the male counterpart. The longer life-span exposes a number of men to the risk of mild-to-severe hypogonadism which in turn we know to be one of the pathogenetic steps toward the loss of bone mineral content in men and in women. Hypogonadism might therefore be one among many corrigible risk factors such as cigarette smoking and alcohol abuse against which clinicians should act in order to prevent osteoporosis and its complications. Treatments with calcium plus vitamin D and bisphophonates are widely used in men, when osteoporosis is documented and hypogonadism has been excluded. The poor knowledge on male osteoporosis accounts for the lack of well shared protocols for the clinical management of the disease. This review focuses on the clinical approach and treatment strategy for osteoporosis in men with particular attention to its relationship with male hypogonadism.

2006 - A novel compound heterozigous mutation of the aromatase gene in an adult man [Abstract in Atti di Convegno]
Laura, Maffei; Rochira, Vincenzo; Evan R., Simpson; Simone, Maria Luisa; Claudio, Aranda; Bibiana, Fabre; M., Stivel; Luberto, Alessandra; Antonio, Balestrieri; Pignatti, Elisa; Paula, Antunez; Carani, Cesare
abstract

Descriptions of a new case of human aromatase deficiency useful for a better understanding of male oestrogen pathophysiology

2006 - A novel compound heterozigous mutation of the aromatase gene in an adult manl [Abstract in Atti di Convegno]
Laura, Maffei; Rochira, Vincenzo; Evan R., Simpson; Simone, Maria Luisa; Claudio, Aranda; Bibiana, Fabre; Mirta, Stivel; Luberto, Alessandra; Antonio, Balestrieri; Pignatti, Elisa; Paula, Antunez; Carani, Cesare
abstract

Descriptions of a new case of human aromatase deficiency useful for a better understanding of male oestrogen pathophysiology

2006 - Carcinoma papillare e Sindrome di Peutz-Jeghers: una rara associazione [Abstract in Atti di Convegno]
Rochira, Vincenzo; Romano, Stefania; Zirilli, Lucia; Madeo, Bruno; Caffagni, Giovanni; Diazzi, Chiara; Valeria, Pugni; Pignatti, Elisa; Roncucci, Luca; PONZ DE LEON, Maurizio; Carani, Cesare; Benatti, Piero
abstract

Case report of a patient with Peutz-Jeghers syndrome and concomitant papillary thyroid cancer that indicates that thyroid cancer might be more frequent in patients with Peutz-Jeghers

2006 - Ferroportin is a monomer in vivo in mice [Articolo su rivista]
Pignatti, Elisa; L., Mascheroni; M., Sabelli; S., Barelli; S., Biffo; Pietrangelo, Antonello
abstract

Ferroportin (FPN) is the main iron export protein in mammals. The actual structure of FPN in vivo and the pathogenesis of ferroportin-related disease are unknown. We aimed at studying the structure and biochemical properties of FPN in mouse tissues that are key for iron homeostasis during various iron manipulations in vivo. We performed glycosylation and oligomerization studies in spleen and liver extracts from mice fed a standard, iron-deprived or iron-enriched diet for 5 months. Purification by affinity chromatography and sucrose gradient show that FPN is not part of a large multiprotein complex. Dietary manipulations did not affect the monomeric status of the native or denatured protein. The glycosylation studies showed that ferroportin is digested by peptide: N-glycosidase F but not by endoglycosidase H. The same results were obtained using protein extracts from iron-deficient or iron-loaded mice. In conclusion, our studies indicate that mouse FPN, regardless of the tissue iron status, is glycosylated but not enriched in mannose residues, and that exists mainly in monomeric form. The latter finding may have important implications for understanding the pathogenesis of the disease due to ferroportin mutations.

2006 - Skeletal effects of estrogen and testosterone in a man with aromatase deficiency: priming effect of estrogen for sex steroids action on bone strength [Abstract in Rivista]
Zirilli, Lucia; Rochira, Vincenzo; Madeo, Bruno; Pignatti, Elisa; Luberto, Alessandra; Caffagni, Giovanni; Diazzi, Chiara; Sanguanini, Alessia; Emile, Roldan; Laura E., Maffei; Carani, Cesare
abstract

The combined treatment with estradiol and testosteron led to optimal parameters of BMD suggesting that testosterone needs estrogens as a permissive factor for a direct androgen anabolic action on bone in men

2005 - Kupffer cells and macrophages are not required for hepatic hepcidin activation during iron overload [Articolo su rivista]
Montosi, Giuliana; Corradini, Elena; Garuti, Cinzia; S., Barelli; S., Recalcati; G., Cairo; L., Valli; Pignatti, Elisa; Vecchi, Chiara; F., Ferrara; Pietrangelo, Antonello
abstract

Hepcidin, the iron hormone, is produced by the liver in response to iron and inflammation. Its synthesis during inflammation is triggered by cytokines, but the details of iron activation are obscure. We tested the role of Kupffer cells and macrophages by studying iron-loaded or inflamed mice with selective inactivation of Kupffer cells or the in vitro effect of conditioned human macrophages on hepcidin expression. Hepcidin messenger RNA (mRNA) expression was studied by Northern blot and reverse transcriptase polymerase chain reaction analysis in mice that were treated with 40 mg/kg gadolinium (III) chloride (GdCl3) as a Kupffer cell inactivating agent and subjected to inflammatory challenges with either lipopolysaccharide (LPS) and turpentine or iron overload by iron-dextran administration. Similar analyses were performed in human hepatoma cells (HepG2) cultured with medium from LPS- or iron-conditioned macrophages from blood donors or patients with HFE-linked hereditary hemochromatosis (HH). In vivo, LPS and particularly turpentine stimulated hepcidin mRNA expression, and this effect was prevented by the inactivation of Kupffer cells. Also, iron overload markedly upregulated hepatic hepcidin mRNA, but this activity persisted in spite of Kupffer cell blockade. In vitro, the medium of LPS-treated normal or hemocromatotic macrophages turned on hepcidin expression. On the contrary, medium of iron-manipulated macrophages, regardless of their HFE status, did not affect hepcidin mRNA steady-state levels. In conclusion, Kupffer cells are required for the activation of hepcidin synthesis during inflammation, and HH inflamed macrophages are capable of mounting a normal response, eventually leading to hepcidin stimulation. However, both Kupffer cells and human macrophages are dispensable for the regulatory activity exerted by iron on hepatic hepcidin.

2005 - Lack of enterocyte iron accumulation in the ferroportin disease [Articolo su rivista]
Corradini, Elena; Montosi, Giuliana; F., Ferrara; A., Caleffi; Pignatti, Elisa; S., Barelli; Garuti, Cinzia; Pietrangelo, Antonello
abstract

Ferroportin-associated iron overload (also known as the ferroportin disease) is a common cause of hereditary hyperferritinemia. It was originally proposed that loss-of-protein function mutations account for iron overload in the FD. This hypothesis is consistent with the 14 phenotype reported in most patients with FD of early iron accumulation in tissues, particularly in macrophages, in spite of relatively normal-low circulatory iron. It was still unclear, however, how FPN mutations would affect iron retention in enterocytes. We studied histologically the intestine of six patients with different FPN mutations as compared to other subjects with various iron disorders. We found that regardless of the underlying FPN mutation, no iron accumulation was found in absorbing enterocytes while, intestinal villi showed marked signs of iron accumulation in the cells of lamina propria. Not surprisingly, in the liver, iron excess was found mainly in Kupffer cells. These results indicate that FPN haploinsufficiency is not limiting for iron export from enterocytes.

2003 - Iron overload in Africans and African-Americans and a common mutation in the SCL40A1 (ferroportin 1) gene [Articolo su rivista]
Gordeuk, Vr; Caleffi, Angela; Corradini, Elena; Ferrara, Francesca; Jones, Ra; Castro, O; Onyekwere, O; Kittles, R; Pignatti, Elisa; Montosi, Giuliana; Garuti, Cinzia; Gangaidzo, It; Gomo, Zar; Moyo, Vm; Rouault, Ta; Macphail, P; Pietrangelo, Antonello
abstract

The product of the SLC40A1 gene, ferroportin 1, is a main iron export protein. Pathogenic mutations in ferroportin 1 lead to an autosomal dominant hereditary iron overload syndrome characterized by high serum ferritin concentration, normal transferrin saturation, iron accumulation predominantly in macrophages, and marginal anemia. Iron overload occurs in both the African and the African-American populations, but a possible genetic basis has not been established. We analyzed the ferroportin 1 gene in 19 unrelated patients from southern Africa (N = 15) and the United States (N = 4) presenting with primary iron overload. We found a new c. 744 C→T (Q248H) mutation in the SLC40A1 gene in 4 of these patients (3 Africans and 1 African-American). Among 22 first degree family members, 10 of whom were Q248H heterozygotes, the mutation was associated with a trend to higher serum ferritin to amino aspartate transferase ratios (means of 14.8 versus 4.3 μg/U; P = 0.1) and lower hemoglobin concentrations (means of 11.8 versus 13.2 g/dL; P = 0.1). The ratio corrects serum ferritin concentration for alcohol-induced hepatocellular damage. We also found heterozygosity for the Q248H mutation in 7 of 51 (14%) southern African community control participants selected because they had a serum ferritin concentration below 400 μg/L and in 5 of 100 (5%) anonymous African-Americans, but we did not find the change in 300 Caucasians with normal iron status and 25 Caucasians with non-HFE iron overload. The hemoglobin concentration was significantly lower in the African community controls with the Q248H mutation than in those without it. We conclude that the Q248H mutation is a common polymorphism in the ferroportin 1 gene in African populations that may be associated with mild anemia and a tendency to iron loading.

2003 - The role of the iron responsive element in the control of ferroportin1/IREG1/MTP1 gene expression [Articolo su rivista]
A., Lymboussaki; Pignatti, Elisa; Montosi, Giuliana; Garuti, Cinzia; Dj, Haile; Pietrangelo, Antonello
abstract

Background/Aims: MTP1/Ferroportin1/IREG1, the product of the SLC40A1 gene, is a main iron export protein in mammals. However, the way this gene is regulated by iron is still unclear. The aim of this study was to investigate the functional role of genomic SLC40A1 elements in response to iron. Methods: Vectors containing either similar to 2.6 kb 5' flanking region or deletion constructs, including one devoid of an iron responsive element (SLC40A1-DeltaIRE-Luc), were analyzed by luciferase reporter gene in transfected HepG2, CaCO2 and U937 cells. Expression of iron genes and activity of the iron regulatory protein were also studied. Results: Iron increased and desferrioxamine decreased luciferase activity in all the cell types using both the full-length construct and the promoter deletion constructs, in the absence of changes in SLC40A1 or luciferase mRNA levels. To test the role of the SLC40A1 5' untranslated region, we first demonstrated that wild type and not SLC40A1-DeltaIRE-Luc could bind iron regulatory protein. Then, in cells transfected with SLC40A1-DIRE-Luc, we found that, in spite of iron regulatory protein activation, the response to iron manipulation was lost. Conclusions: We demonstrate that the iron responsive element in the SLC40A1 gene is functional and that it controls gene expression through the cytoplasmic iron regulatory protein system. (C) 2003 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

2001 - Autosomal-dominant hemochromatosis is associated with a mutation in the ferroportin (SLC11A3) gene [Articolo su rivista]
Montosi, Giuliana; Donovan, A.; Totaro, Antonella; Garuti, Cinzia; Pignatti, Elisa; Cassanelli, S.; Trenor, C. C.; Gasparini, P.; Andrews, N. C.; Pietrangelo, Antonello
abstract

Hemochromatosis is a progressive iron overload disorder that is prevalent among individuals of European descent. It is usually inherited in an autosomal-recessive pattern and associated with missense mutations in HFE, an atypical major histocompatibility class I gene. Recently, we described a large family with autosomal-dominant hemochromatosis not linked to HFE and distinguished by early iron accumulation in reticuloendothelial cells. Through analysis of a large pedigree, we have determined that this disease maps to 2q32. The gene encoding ferroportin (SLC11A3), a transmembrane iron export protein, lies within a candidate interval defined by highly significant lod scores. We show that the iron-loading phenotype in autosomal-dominant hemochromatosis is associated with a nonconservative missense mutation in the ferroportin gene. This missense mutation, converting alanine to aspartic acid at residue 77 (A77D), was not seen in samples from 100 unaffected control individuals. We propose that partial loss of ferroportin function leads to an imbalance in iron distribution and a consequent increase in tissue iron accumulation.

2001 - Frequency and biochemical expression of C282Y/H63D hemochromatosis (HFE) gene mutations in the healthy adult population in Italy [Articolo su rivista]
Cassanelli, S; Pignatti, Elisa; Montosi, Giuliana; Garuti, Cinzia; Mariano, M; Campioli, D; Carbonieri, A; Baldini, Enrica; Pietrangelo, Antonello
abstract

Background/Aims: The actual prevalence of the main hemochromatosis (HFE) mutations in the Italian adult population and their phenotypic expression have not yet been established. This information is key to advocate a mass-screening program. Methods: Two thousand one hundred adults were tested for the C282Y/H63D HFE gene mutations by an automated genotyping assay as well as transferrin saturation (TS) and serum ferritin levels. Results: No homozygotes for the C282Y mutation were found. Heterozygosity for the C282Y mutation was 3.1%, while heterozygosity and homozygosity for the H63D mutation were 21.5% and 2.5%, respectively. TS was significantly higher in C282Y heterozygotes and H63D homozygotes as compared to wild-type individuals (P < 0.01). Interestingly, of the HFE wild-type subjects 5.9% had a TS value above the 45% threshold. Conclusions: This study shows that (i) the predicted prevalence for C282Y homozygosity in Italy is 1:3900; (ii) the C282Y/H63D wild-type population has an increased baseline of iron parameters possibly due to genetic factors not linked to the C282Y/H63D mutations; (iii) since in the latter population the actual tissue iron burden cannot be assessed, phenotypic (TS) screening in Italy is not recommended until the true prevalence of all mutations in the HFE gene and in other hemochromatosis genes will be established. (C) 2001 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved.