Functional characterization of a CDKN1B mutation in a Sardinian kindred with multiple endocrine neoplasia type 4 (MEN4)

Inactivating germline mutations of the CDKN1B gene, encoding for the nuclear cyclin-dependent kinase inhibitor p27kip1 protein, have been reported in patients with multiple endocrine neoplasia type 4 (MEN4), a MEN1-like phenotype without MEN1 mutations. The aim of this study was to in vitro characterize the germline CDKN1B mutation c.374_375delCT (S125X) we detected in a patient with MEN4. The proband was affected by multiglandular primary hyperparathyroidism and gastro-entero-pancreatic tumors. We carried out subcellular localization experiments transfecting into eukaryotic HeLa and GH3 cell lines plasmid vectors expressing the CDKN1B wild type (wt) or mutant cDNA. Western blot studies showed that fusion proteins were expressed at equal levels. The mutated protein was shorter compared to the wt protein and lacked the highly conserved C-terminal domain, which includes the bipartite nuclear localization signal at amino acids 152/153 and 166/168. In HeLa and GH3 cells wt p27 localized in the nucleus whereas the p27_S125X protein was retained in the cytoplasm predicting the loss of tumor suppressive function. The proband's tumoral parathyroid tissue did not show allelic loss, since wt and mutant alleles were both present by sequencing the somatic DNA. Immunohistochemistry showed a complete loss of nuclear p27 expression in the parathyroid adenoma removed by the patient at the second surgery. In conclusion, our study confirms the pathogenic role of the c.374_375delCT CDKN1B germline mutation in a patient with MEN4

Quantitative evaluation of healthy epidermis by means of multiphoton microscopy and fluorescence lifetime imaging microscopy

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Mutational and large deletion study of genes implicated in hereditary forms of primary hyperparathyroidism and correlation with clinical features

The aim of this study was to carry out genetic screening of the MEN1, CDKN1B and AIP genes, both by direct sequencing of the coding region and multiplex ligation-dependent probe amplification (MLPA) assay in the largest monocentric series of Italian patients with Multiple Endocrine Neoplasia type 1 syndrome (MEN1) and Familial Isolated Hyperparathyroidism (FIHP). The study also aimed to describe and compare the clinical features of MEN1 mutation-negative and mutation-positive patients during long-term follow-up and to correlate the specific types and locations of MEN1 gene mutations with onset and aggressiveness of the main MEN1 manifestations. A total of 69 index cases followed at the Endocrinology Unit in Pisa over a period of 19 years, including 54 MEN1 and 15 FIHP kindreds were enrolled. Seven index cases with MEN1 but MEN1 mutation-negative, followed at the University Hospital of Cagliari, were also investigated. FIHP were also tested for CDC73 and CaSR gene alterations. MEN1 germline mutations were identified in 90% of the index cases of familial MEN1 (F-MEN1) and in 23% of sporadic cases (S-MEN1). MEN1 and CDC73 mutations accounted for 13% and 7% of the FIHP cohort, respectively. A CDKN1B mutation was identified in one F-MEN1. Two AIP variants of unknown significance were detected in two MEN1-negative S-MEN1. A MEN1 positive test best predicted the onset of all three major MEN1-related manifestations or parathyroid and gastro-entero-pancreatic tumors during follow-up. A comparison between the clinical characteristics of F and S-MEN1 showed a higher prevalence of a single parathyroid disease and pituitary tumors in sporadic compared to familial MEN1 patients. No significant correlation was found between the type and location of MEN1 mutations and the clinical phenotype. Since all MEN1 mutation-positive sporadic patients had a phenotype resembling that of familial MEN1 (multiglandular parathyroid hyperplasia, a prevalence of gastro-entero-pancreatic tumors and/or the classic triad) we might hypothesize that a subset of the sporadic MEN1 mutation-negative patients could represent an incidental coexistence of sporadic primary hyperparathyroidism and pituitary tumors or a MEN1 phenocopy, in our cohort, as in most cases described in the literature

Should parafibromin staining replace HRTP2 gene analysis as an additional tool for histologic diagnosis of parathyroid carcinoma?

Objective: HRPT2 gene mutations are associated with parathyroid carcinomas, and absence of parafibromin immunoreactivity has been suggested as a diagnostic marker of malignancy. The aim of our study was to extend parafibromin studies in a series of benign and malignant parathyroid tumors and cross-validate the results of immunohistochemistry with those of HRPT2 analysis. Design and patients: We performed parafibromin and cyclin D1 immunostaining and HRPT2 gene analysis using loss of heterozygosity studies and sequencing analysis in parathyroid specimens from 11 patients with carcinoma (eleven primary tumors, one skin, and four lung metastases), 22 with sporadic adenomas, and 4 with atypical adenomas. Results: Ten out of eleven parathyroid cancers were negative for parafibromin staining and showed HRPT2 gene abnormalities. The remaining sample was negative for immunostaining and genetic analyses. All but one sporadic adenomas showed parafibromin immunoreactivity and no HRPT2 gene abnormalities. The sample with negative immunostaining carried an HRPT2 mutation. Two atypical adenomas were positive and two negative with parafibromin staining. No HRPT2 abnormalities were found in these samples. Cyclin D1 expression was heterogeneous and there was no relationship between expression/expression level of cyclin D1 and parafibromin expression. Conclusions: We have shown that negative parafibromin staining is almost invariably associated with HRPT2 mutations and confirm that loss of parafibromin staining strongly predicts parathyroid malignancy. In clinical practice, these tests could be particularly useful in the subset of parathyroid tumors with equivocal histological examination. However, their diagnostic value in this setting remains to be proven

Identification and functional characterization of loss-of-function mutations of the calcium-sensing receptor in four italian kindreds with familial hypocalciuric hypercalcemia

Objective: Identification and characterization of calcium-sensing receptor (CASR) mutations in four unrelated Italian kindreds with familial hypocalciuric hypercalcemia. Design: Clinical evaluation and genetic analysis of CASR gene. Functional characterization of mutated CASRs. Methods: Direct sequencing of CASR gene in genomic DNA. Studies of CASR-mediated increases in cytosolic calcium concentration [Ca2C]i in CASR-transfected COS-7 cells in vitro. Results: Four unreported heterozygous CASR mutations were identified, including three missense (H595Y, P748H, and C765W) and one splice site (IVS2C1GOC) mutation. The H595Y, P748H, and C765W mutant receptors, although expressed at normal levels on the cell surface, showed a reduced response in [Ca2C]i relative to the wildtype (WT) CASR to increasing extracellular calcium concentrations. Cotransfection experiments showed that the H595Y and P748H mutants did not affect the apparent affinity of the WT CASR for calcium, suggesting that they do not exert a dominant-negative effect. On the other hand, the co-transfected C765W mutant decreased the maximum response of the WT CASR to calcium, suggesting that it may reduce the effective concentration of the normal CASR on the cell surface or impair its maximal signaling capacity. Conclusions: Four CASR mutations were identified. The reduced functional responses to extracellular calcium and normal expression of the mutant receptors suggest that conformational changes account for altered CASR activity. Moreover, a reduced complement of normal CASRs in these heterozygous patients, perhaps combined with a mutant receptor-induced decrease in maximal activity of the WT receptor, may contribute to defective calcium-sensing in vivo.L'articolo è disponibile sul sito dell'editore http://www.euro-endo.org/default.asp

Case report: Early-onset osteoporosis in a patient carrying a novel heterozygous variant of the WNT1 gene

Article presents a case study of a 35 year-old Caucasian woman who experienced multiple vertebral fractures two months after her second pregnancy. The results suggest a potential role of heterozygous WNT1 variants in the pathogenesis of early-onset osteoporosis. The authors suggest that teriparatide is one of the most appropriate available therapies for such cases

Cambiamento organizzativo e tecnologie it abilitanti: analisi di un caso aziendale nel settore meccanico

L’elaborato proposto analizza le fasi del cambiamento in seguito all’introduzione del modulo per la gestione del magazzino del sistema informativo Business Net all’interno dell’officina meccanica Ghisellini Giuseppe, una piccola azienda operante nel settore dell’artigianato con una competenza maturata negli anni nel settore delle pompe e dei motori idraulici e dei motori marini. In seguito ad un’analisi delle forze resistenti e delle spinte al cambia-mento, viene ricercata la causa delle inefficienze del sistema basandosi sul metodo delle otto fasi proposto da Kotter. Dal momento che l’inefficienza maggiore viene ritrovata nella scarsa circolazione di informazioni, si ritiene necessario una riprogettazione dei processi aziendali, attraverso una mappatura dei processi “as is” e “to be”, in cui viene proposta una soluzione che permette una maggiore circolazione delle informazioni. Tale soluzione proposta è infine valutata economicamente

Ruolo del gene CDKN1B/p27Kip1 nella tumorigenesi paratiroidea

RIASSUNTO La forma sporadica di iperparatiroidismo primario (PHPT) è la più comune, mentre le forme ereditarie sono relativamente rare e comprendono: la sindrome delle Neoplasie Endocrine Multiple di tipo 1 (MEN1), di tipo 2 (MEN2A), di tipo 4 (MEN4), l’Iperparatiroidismo associato a tumori della mandibola (HPT-JT), l’Ipercalcemia Ipocalciurica Familiare (FHH) e l’Iperparatiroidismo Familiare Isolato (FIHP). In alcuni casi il PHPT è causato da un adenoma atipico che ha diversi aspetti istologici tipici del tumore maligno ma non presenta invasione capsulare o vascolare, né metastasi a distanza. Nonostante mutazioni inattivanti del gene oncosoppressore MEN1 siano presenti in circa il 20-35% dei casi di adenoma sporadico, in >80% di MEN1 familiare, in circa il 30% di MEN1 sporadica ed in circa il 25% di famiglie FIHP, resta un’alta percentuale di casi con patogenesi molecolare sconosciuta. La menina, codificata dal gene MEN1, è coinvolta nel controllo del ciclo cellulare attraverso la sua interazione con il complesso MLL (Mixed LineageLeukemia), che promuove l’attivazione trascrizionale dell’inibitore chinasicociclina-dipendente p27Kip1, un importanteregolatore del ciclo cellulare. Mutazioni germinali inattivanti del gene CDKN1B (12p13) sono responsabili della sindrome MEN4. In circa il 3% degli adenomi sporadici, analizzati fino ad ora in letteratura, è presente una mutazione del gene CDKN1B. Lo scopo di questo studio è stato quello di indagare la presenza di mutazioni del gene CDKN1B in un ampio gruppo di adenomi sporadici (147), in 10 adenomi atipici, in 21 casi affetti da sindrome MEN1 ma negativi all’analisi mutazionale del gene MEN1 ed in 15 famiglie FIHP negative all’analisi mutazionale dei geni MEN1, CDC73 e CASR. In un sottogruppo di 50 adenomi sporadici e negli adenomi atipici sono state anche indagate l’espressione di p27Kip1, con l’analisi immunoistochimica, ed è stata valutata la correlazione tra la ridotta espressione di p27Kip1 e l’eventuale presenza di mutazioni somatiche del gene MEN1. Tre varianti del gene CDKN1B (c.-80C>T, c.-29_-26delAGAG, p.P133T) sono state identificate, a livello germinale, in 3/147 adenomi sporadici, mentre nessuna variante è stata individuata negli adenomi atipici e nei casi affetti dai disordini ereditari MEN1 e FIHP. Lo studio in vitro ha dimostrato che la nuova variante c.-80C>T determinava una significativa riduzione del tasso di trascrizione di CDKN1B. All’immunoistochimica l’adenoma con la delezione c.-29_-26delAGAG mostrava una completa perdita di espressione di p27Kip1ed una notevole riduzione della quantità di mRNA. Due dei tumori portatori della mutazione CDKN1B avevano anche una mutazione a livello del gene MEN1. Il 54% dei tumori risultati negativi all’analisi mutazionale di CDKN1B avevano una riduzione di espressione di p27Kip1 a livello nucleare. La ridotta espressione a livello nucleare e citoplasmatico di p27Kip1 è risultata associata in maniera statisticamente significativa alla presenza di mutazioni somatiche del gene MEN1, identificate in 15/50 dei campioni, suggerendo la de-regolazione del meccanismo di attivazione della trascrizione di CDKN1Bmenina-mediato. Tutti gli adenomi atipici analizzati presentavano perdita di espressione di p27Kip1 a livello nucleare. A differenza di quanto osservato per gli adenomi, l’incidenza delle mutazioni di MEN1 (1/10) sembra avere un ruolo minore nella regolazione del p27Kip1. In conclusione, questo studio documenta un’elevata incidenza di casi di adenoma tipico e atipico di natura sporadica con una ridotta o totale perdita di espressione di p27Kip1, che non risulta associata a mutazioni del gene CDKN1B, ma suggerisce che la regolazione della trascrizione di CDKN1B, mediata dalla menina, possa avere un ruolo nella tumorigenesi paratiroidea. L’assenza di mutazioni del gene CDKN1B nella nostra casistica di forme sindromiche (MEN1) e non (FIHP) di iperparatiroidismo ereditario conferma la rarità dei casi MEN4 descritti in letteratura. Anche mutazioni somatiche del gene CDKN1B sembrano essere particolarmente rare nei casi di adenoma e adenoma atipico sporadici. ABSTRACT Primary HyperParaThyroidism (PHPT) is usually a sporadic disorder while familial syndromes are relatively rare and includes: Multiple Endocrine Neoplasia type 1 (MEN1), type 2 (MEN2A) and type 4 (MEN4), HyperParaThyroidism-Jaw Tumor syndrome (HPT-JT), Familial Hypocalciuric Hypercalcemia (FHH) and Familial Isolated HyperParathyroidism (FIHP). PHPT can also be caused by atypical parathyroid adenomas that represent a subset of tumors with histological features worrisome for carcinoma without capsular and vascular invasion and metastasis. Although activating mutations of the MEN1 tumor suppressor gene (11q13) are present in approximately 20-35% of cases of sporadic adenoma, in>80% of MEN1 families, in about 30% of sporadic MEN1 and in about 25% of FIHP index cases, it remains a high percentage of cases with unknown molecular pathogenesis. Menin, encoded by the MEN1 gene, is involved in cell cycle control through its interaction with the complex MLL (Mixed Lineage Leukemia) that promotes transcriptional activation of the cyclin-dependent kinase inhibitor p27Kip1, an important regulator of the cell cycle.Recently, inactivating germline mutations ofCDKN1B gene have been detected in about 3% of sporadic parathyroid adenomas. In the present study we further investigate the pathogenic role of the CDKN1B gene in a large series of sporadic parathyroid adenomas (147), 10 atypical adenomas, 21MEN1 patients which were negative to the MEN1 genetic screening together with 15 FIHPkindreds, negative in a previous mutational analysis of MEN1, CDC73 and CASR genes. The relationship between p27Kip1immunohistochemical staining and the MEN1 mutational status were also performed in a subgroup of 50 CDKN1B mutation-negative parathyroid adenomas and in all the atypical adenomas. Three germline CDKN1B variants (c.-80C>T, c.-29_-26delAGAG, c.397C>A) were identified in 3/147 patients. No mutation was detected in the atypical adenomas and in the syndromic and non-syndromic form of PHPT. Reduction of CDKN1B gene transcription rate was demonstrated in vitro for the novel c.-80C>T and the c.-29_-26delAGAG variants. Loss of p27Kip1expression and a significant mRNA reduction were detected in the tumor carrying the c.-29_-26delAGAG variant. Two tumors carrying the CDKN1B variants also harbored a MEN1 mutation. Fifty-four percent of 50 CDKN1B mutation-negative tumors had a reduction of p27Kip1 nuclear staining. Somatic MEN1 mutations, identified in 15/50samples, significantly segregated in tumors negative for nuclear and cytoplasmic p27 staining, suggesting a transcriptional dysregulation of p27Kip1 menin-mediated. Loss p27Kip1 nuclear expression was detected in all atypical adenomas. The incidence of MEN1 mutations (1/10) seems to have a minor role in the regulation of p27Kip1 respect to typical adenomas. In conclusion, this study reveals a high rate of downregulation of the p27 levels in the sporadic typical and atypical adenomas in spite of the lack of somatic CDKN1B mutations, and suggests a role for menin in transcriptional regulation of CDKN1B during parathyroid tumorigenesis, as demonstrated by murine models. The lack of CDKN1B mutations in our group of syndromic (MEN1) and non-syndromic (FIHP) hereditary hyperparathyroidism confirm the paucity of MEN4 cases in the literature. Nonetheless, CDKN1B somatic mutations seems to be very rare both in sporadic typical and atypical adenomas

Parathyroid tumorigenesis

Primary hyperparathyroidism (PHPT) is a common endocrinopathy, mostly caused by a monoclonal parathyroid adenoma. This review primarily summarizes current knowledge concerning molecular pathogenesis of familial forms of primary hyperparathyroidism and sporadic (non familial) parathyroid tumors. The hereditary syndromes have been recognized as exhibiting Mendelian inheritance patterns and include multiple endocrine neoplasia types 1 (MEN 1) and 2A (MEN 2A), hereditary hyperparathyroidism- jaw tumor (HPT-JT) syndrome, familial isolated hyperparathyroidism (FIHP), familial hypocalciuric hypercalcemia (FHH) and severe neonatal hyperparathyroidism (NSHPT). Inactivating mutations of MEN1 tumor suppressor gene are responsible for MEN 1 in >90% of cases. MEN1 gene has also an established role in the pathogenesis of sporadic parathyroid adenomas. Allelic loss (LOH) of chromosome 11q13 occurs in about 30-40% and somatic mutation of MEN1 gene occur in about 12-20% of sporadic parathyroid adenomas. A mouse model of MEN1 deficiency causes a phenotype that includes the same range of major endocrine tumors as in MEN 1 patients, and exhibits multistage tumor progression with metastatic potential. Hormonal disturbances, such as abnormal PTH and insulin levels, were also observed in these mice. Mutations in a newly identified tumor suppressor gene, HRPT2, have been recently associated with the development of HPT-JT. HRPT2 mutations are also frequent in sporadic parathyroid carcinomas and central to their pathogenesis. MEN1 and HRPT2 genes mutations have also been found in a subset of FIHP families. FHH and NSHPT represent the mildest and severest variants of PHPT, respectively. Both cause hypercalcemia from birth and atypical PHPT that always uniquely persists after subtotal parathyroidectomy. Future identification of additional oncogenes and tumor suppressor genes will clarify the molecular basis of abnormalities of parathyroid proliferation and regulatory function and other specific features unique to the parathyroid tumorigenesis