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C Asteria, L Persani, M Ferrari and P Beck-Peccoz

This study analyzed the structural differences of the carbohydrate chains of circulating free alpha-submit (alpha-SU) hypersecreted in various non-tumoral (primary hypothyroids, postmenopausal women, patients with chronic uremia, normal fetuses) and tumoral (gut carcinoids, TSH-, GH- and pure alpha-secreting pituitary adenomas) clinical conditions. Carbohydrate structures of free alpha-SU were investigated by means of lectin affinity chromatography using Concanavalin A (Con-A), which allows the separation of free alpha-SU in three different fractions (unbound = UB, weakly bound = WB and firmly bound = FB) depending on the nature and maturation of glycosylated chains. The concentrations of alpha-SU in serum and in Con-A fractions were measured by a sensitive and specific IRMA. Free alpha-SU hypersecreted from postmenopausal women, primary hypothyroids, and patients with chronic uremia showed similar binding patterns to Con-A, the percentage of UB fractions (UB: 44.5 +/- 1.9%, 39.5 +/- 3.8%, 48.2 +/- 5.6% respectively) being higher than both WB and FB fractions (WB: 33.2 +/- 1.4%, 30.7 +/- 4.6%, 28.5 +/- 2.1%; FB: 22.3 +/- 0.7%, 29.8 +/- 6.6%, 23.3 +/- 4.2% respectively). In normal fetuses the amount of UB fraction was very high (UB: 70.7 +/- 5.4%). Free alpha-SU from patients with TSH- and GH-secreting adenomas showed a binding pattern to Con-A significantly different from that observed in postmenopausal women taken as controls, the WB fractions being significantly higher (WB: 56.9 +/- 16.8% and 71 +/- 12.4% respectively, P < 0.001). A typical pattern of elution on Con-A, characterized by a prevalence of immature alpha-SU molecules eluted in the FB fraction, was found in patients with pure alpha-secreting adenomas. This chromatographic behavior was significantly different from that seen in the controls, as well as in other pituitary tumors and in gut carcinoids (FB: 41.8 +/- 5.0%, 22.3 +/- 0.7%, 16.8 +/- 6.6%, 10.6 +/- 2.0% respectively). Moreover, in these latter patients the pattern of free alpha-SU binding was exactly the opposite of that observed in pure alpha-secreting adenomas, with a prevalence of mature alpha-SU molecules (UB: 59.1 +/- 4.4 vs 18.3 +/- 7.2%). In conclusion, our data on Con-A affinity chromatography clearly demonstrate that carbohydrate branching of circulating free alpha-SU varies in patients with pituitary adenomas as compared with patients with gut carcinoids or other non-tumoral conditions. Moreover, the finding of a greater proportion of circulating free alpha-SU forms that firmly bind to Con-A in patients with pure alpha-secreting adenomas, seems to be pathognomonic of non-functioning pituitary adenomas.

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L Alberti, MC Proverbio, S Costagliola, G Weber, P Beck-Peccoz, G Chiumello and L Persani

OBJECTIVE: Clinical and genetic investigations were undertaken in a case of familial hyperthyroidism, with onset of thyrotoxic symptoms varying between childhood/adolescence. METHODS: Automatic sequence analysis was carried out of the TSH receptor (TSHR) gene. Functional studies were undertaken of mutant TSHR in transient expression experiments in COS-7 cells including the evaluation of cAMP accumulation and of protein expression by flow cytometry, as well as the calculation of specific constitutive activity (SCA). RESULTS: In four affected cases, the age of onset of thyrotoxic manifestations of non-autoimmune origin varied between 5 and 18 years. The disease transmission was typically autosomal dominant. TSHR gene sequence revealed the presence of a germline heterozygous substitution at codon 597 leading to the novel mutation V597F. This residue is located in the 5th transmembrane domain of the receptor protein in a critical region for membrane targeting and signal transduction. Functional studies of the V597F mutant indicate an 11-fold increase in SCA, associated with a reduction in receptor protein expression on the cytoplasmic membrane. CONCLUSIONS: Description was made of a family with non-autoimmune autosomal dominant hyperthyroidism carrying a novel mutation of TSHR leading to the increment in specific constitutive activity. Factors that may influence the clinical expression of TSHR germline mutations are discussed.

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MC Ferrari, R Parini, MD Di Rocco, G Radetti, P Beck-Peccoz and L Persani

OBJECTIVE: The congenital disorders of glycosylation (CDGs) are progressive multisystemic disorders characterized by a heterogeneous deficiency of the carbohydrate moieties in various structural and circulating glycoproteins, representing a natural model for glycoprotein hormone studies. Here, we studied the carbohydrate moiety of circulating glycoprotein hormones in four patients with a clinical suspicion of CDGs. METHODS: The diagnosis of CDG-I was confirmed in two out of the four cases by transferrin isoelectrofocusing (IEF) and/or carbohydrate-deficient transferrin (CDT) test. The carbohydrate moiety of serum endocrine-related glycoproteins was investigated by means of Ricin (immunopurified thyrotropin (TSH)) and Concanavalin A (Con-A) (TSH, follicle-stimulating hormone, alpha-subunit and thyroglobulin) lectin affinity chromatography measurement. RESULTS: CDT concentrations were very high in the two patients with CDG-I and moderately enhanced in the remaining two. In the two CDG-I patients, Ricin analysis of immunopurified TSH showed a severe impairment of lectin binding, both before and after neuroaminidase treatment, indicating a nearly complete lack of terminal sialic acid and galactose residues. In these two cases, Con-A analysis showed a significant prevalence of firmly bound isoforms with poorly processed carbohydrate chains. In the remaining two cases with unknown CDG classification, TSH binding pattern to Ricin was modestly affected and Con-A analysis showed the prevalence of weakly bound glycoprotein isoforms. CONCLUSIONS: The results of Ricin analyses in all four patients were consistent with the CDT test and/or serum transferrin IEF. The severe alteration of TSH binding pattern to Ricin seems to be characteristic of CDG-I. Nevertheless, TSH biological properties are not severely altered, as normal thyroid function was found in both cases.

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S Corbetta, P Englaro, S Giambona, L Persani, WF Blum and P Beck-Peccoz

Leptin is the protein product of the ob gene, secreted by adipocytes. It has been suggested that it may play an important role in regulating appetite and energy expenditure. The aim of this study was to evaluate a possible interaction of thyroid hormones with the leptin system. We studied 114 adult patients (65 females and 49 males): 36 were affected with primary hypothyroidism (PH), 38 with central hypothyroidism (CH) and 40 with thyrotoxicosis (TT). Patients with CH were studied both before and after 6 months of L-thyroxine replacement therapy. Body mass index (BMI; kg/m2), thyroid function and fasting serum leptin were assessed in all patients. Since BMI has been proved to be the major influencing variable of circulating leptin levels, data were expressed as standard deviation score (SDS) calculated from 393 male and 561 female controls matched for age and BMI. No difference in SDS was recorded between males and females whatever the levels of circulating thyroid hormones. In males, no significant difference was recorded among the SDSs of PH (-0.36 +/- 1.2), TT (-0.35 +/- 1.2) and CH (0.01 +/- 1.4) patients. Females with PH had an SDSs significantly lower than TT females (-0.77 +/- 1.0 vs -0.06 +/- 1.2; P < 0.02), while no significant differences between CH (-0.34 +/- 0.7) and TT females or between CH and PH females were observed. SDS in CH patients after 6 months of L-thyroxine therapy significantly varied only in females (0.25 +/- 1.4). In conclusion, circulating thyroid hormones do not appear to play any relevant role in leptin synthesis and secretion. However, as females with either overt hypo- or hyper-thyroidism or central hypothyroidism after L-thyroxine therapy show differences in their SDSs, a subtle interaction between sex steroids and thyroid status in modulating leptin secretion, at least in women, may occur.

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A Lania, E Ballare, S Corbetta, M Filopanti, L Persani and A Spada

OBJECTIVE: The GH-releasing peptide GHRP-6, has been found to interact with specific receptors in somatotrophs, causing cytosolic Ca2+ ([Ca2+]i) rise and GH release. Moreover, this peptide has been demonstrated to stimulate the secretion of pituitary hormones other than GH, i.e. ACTH and prolactin, this effect being generally attributed to a central action. In this study we evaluated whether the pituitary action of this peptide is restricted to cell type of somatotroph lineage. METHODS: The effect opf GHRP-6 on [Ca2+]i was tested in cell preparations obtained from a series of human pituitary adenomas (9 GH-secreting adenomas, 7 nonfunctioning adenomas, 3 ACTH-secreting adenomas, 2 TSH-secreting adenomas and 1 prolactinoma) loaded with the Ca2+ indicator fura-2. RESULTS: GHRP-6, at concentrations higher than 1 nmol/l, significantly increased [Ca2+]i in all tumours, with the exception of the 3 ACTH-secreting adenomas in which the peptide was ineffective at any concentration tested (from 1 nmol/l to 1 micromol/l). By contrast, in all ACTH-secreting adenomas, both corticotrophin-releasing hormone and pituitary adenylate cyclase activating peptide caused a marked [Ca2+]i increase. In tumours responsive to GHRP-6, the peptide caused a typical biphasic [Ca2+]i rise due to Ca2+ mobilization from the intracellular stores and Ca2+ influx through voltage-dependent Ca2+ channels. CONCLUSIONS: These data indicate that almost all tumoral pituitary cell types are targets of GHRP-6 action, the only exception being corticotrophs.

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S R Ali, J Bryce, M Cools, M Korbonits, J G Beun, D Taruscio, T Danne, M Dattani, O M Dekkers, A Linglart, I Netchine, A Nordenstrom, A Patocs, L Persani, N Reisch, A Smyth, Z Sumnik, W E Visser, O Hiort, A M Pereira, S F Ahmed and on behalf of Endo-ERN

Objective

To identify cross-border international registries for rare endocrine conditions that are led from Europe and to understand the extent of engagement with these registries within a network of reference centres (RCs) for rare endocrine conditions.

Methods

Database search of international registries and a survey of RCs in the European Reference Network for rare endocrine conditions (Endo-ERN) with an overall response rate of 82%.

Results

Of the 42 conditions with orphacodes currently covered within Endo-ERN, international registries exist for 32 (76%). Of 27 registries identified in the Orphanet and RD-Connect databases, Endo-ERN RCs were aware of 11 (41%). Of 21 registries identified by the RC, RD-Connect and Orphanet did not have a record of 10 (48%). Of the 29 glucose RCs, the awareness and participation rate in an international registry was highest for rare diabetes at 75 and 56% respectively. Of the 37 sex development RCs, the corresponding rates were highest for disorders of sex development at 70 and 52%. Of the 33 adrenal RCs, the rates were highest for adrenocortical tumours at 68 and 43%. Of the 43 pituitary RCs, the rates were highest for pituitary adenomas at 43 and 29%. Of the 31 genetic tumour RCs, the rates were highest for MEN1 at 26 and 9%. For the remaining conditions, awareness and participation in registries was less than 25%.

Conclusion

Although there is a need to develop new registries for rare endocrine conditions, there is a more immediate need to improve the awareness and participation in existing registries.

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V Estienne, C Duthoit, VD Costanzo, PJ Lejeune, M Rotondi, S Kornfeld, R Finke, JH Lazarus, U Feldt-Rasmussen, WG Franke, P Smyth, M D'Herbomez, B Conte-Devolx, L Persani, C Carella, Jourdain JR, M Izembart, ME Toubert, A Pinchera, A Weetman, R Sapin, P Carayon and J Ruf

OBJECTIVE: TGPO autoantibodies (aAbs) that bind simultaneously to thyroglobulin (Tg) and thyroperoxidase (TPO) are present in the serum of patients with autoimmune thyroid diseases (AITD) and have been found to differ from monospecific Tg and TPO aAbs. To obtain further insights on the prevalence defined as the rate of occurrence and significance of TGPO aAbs in a large population, we carried out a collaborative study involving 15 European teams. METHODS: Serum samples from 3122 patients with various thyroid and non-thyroid diseases and normal subjects were assayed using a novel TGPO aAb detection kit. This test was designed so that TGPO aAbs are trapped between the Tg-coated solid phase and the soluble TPO labeled with a radioiodinated monoclonal antibody. RESULTS: Only three out of the 220 normal subjects (prevalence of 1.4%) were found to have positive TGPO aAb levels, which were mainly observed in the patients with AITD: the group of patients suffering from Hashimoto's thyroiditis had a TGPO aAb prevalence of 40.5% (n=437 patients), those with Graves' disease, a prevalence of 34.6% (n=645) and those with post-partum thyroiditis, 16.0% (n=243). Among the non-AITD patients with positive TGPO aAb levels, the TGPO aAb prevalence ranged from 20.7% among those with thyroid cancer (n=246) to 0% among those with toxic thyroid nodules (n=47). Among the patients with non-thyroid diseases, the TGPO aAb prevalence ranged from 9.8% in the case of Biermer's pernicious anemia (n=78) to 0% in that of premature ovarian failure (n=44). It is worth noting that the groups showing the highest TGPO aAb prevalence also contained the patients with the highest TGPO aAb titers. Statistical comparisons between the TGPO aAb prevalences in the various groups showed that TGPO aAb could be used as a parameter to distinguish between the groups of Hashimoto's and Graves' patients and between the women with post-partum thyroiditis and the post-partum women with only Tg and/or TPO aAb established during early pregnancy. Unexpectedly, the correlations between TGPO aAbs and Tg and TPO aAbs were found to depend mainly on the assay kit used. CONCLUSION: High TGPO aAb titers are consistently associated with AITD but the reverse was not found to be true. TGPO aAbs are a potentially useful tool, however, for establishing Hashimoto's diagnosis, and would be worth testing in this respect with a view to using them for routine AITD investigations.

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Marco Bonomi, Valeria Vezzoli, Csilla Krausz, Fabiana Guizzardi, Silvia Vezzani, Manuela Simoni, Ivan Bassi, Paolo Duminuco, Natascia Di Iorgi, Claudia Giavoli, Alessandro Pizzocaro, Gianni Russo, Mirella Moro, Letizia Fatti, Alberto Ferlin, Laura Mazzanti, Maria Chiara Zatelli, Salvo Cannavò, Andrea M Isidori, Angela Ida Pincelli, Flavia Prodam, Antonio Mancini, Paolo Limone, Maria Laura Tanda, Rossella Gaudino, Mariacarolina Salerno, Pregnolato Francesca, Mohamad Maghnie, Mario Maggi, Luca Persani and Italian Network on Central Hypogonadism

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 non-reproductive 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 classified 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 significantly 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 significantly 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 findings improve the understanding of IHH and may have a positive impact on the management of patients and their families.