Context Measurement of circulating microRNAs (miRNAs) as potential biomarkers of fragility fracture risk has recently become a subject of investigation. Objective Measure by next-generation sequencing (NGS), global miRNA expression in serum samples of osteoporotic subjects vs individuals with normal bone mineral density (BMD). Design Samples were collected from patients with different bone phenotypes and/or fragility fractures who did not receive any antiresorptive and/or bone-forming drug at the time of blood collection. Setting Samples and data were collected at 7 medical centers in Italy. Patients NGS prescreening: 50 osteoporotic patients vs 30 individuals with normal BMD. Droplet digital polymerase chain reaction (ddPCR) validation: 213 patients with different bone phenotypes, including the NGS-analyzed cohort. Results NGS identified 5 miRNAs (miR-8085, miR-320a-3p, miR-23a-3p, miR-4497, miR-145-5p) differentially expressed in osteoporosis cases without fractures vs controls. ddPCR validation confirmed lower c-miR-23a-3p expression in osteoporotic patients, with or without fracture, than in osteopenic and normal subjects and increased c-miR-320a-3p expression in osteoporotic patients with fracture and lower expression in osteoporotic patients without fracture. ddPCR analysis showed a significantly increased expression of miR-21-5p in osteoporotic patients, with or without fracture, than in osteopenic and normal subjects, not evidenced by the NGS prescreening. Discussion Our study confirmed levels of c-miR-23a-3p and c-miR-21-5p as able to distinguish osteoporotic patients and subjects with normal BMD. Increased levels of c-miR-320a-3p specifically associated with fractures, independently by BMD, suggesting c-miR-320a-3p as a prognostic indicator of fracture risk in osteoporotic patients, to be confirmed in prospective studies on incident fractures.

The discovery that osteocytes secrete phosphaturic fibroblast growth factor 23 (FGF23) has defined bone as an endocrine organ. However, the autocrine and paracrine functions of FGF23 are still unknown. The present study focuses on the cellular and molecular mechanisms involved in the complex control of FGF23 production and local bone remodeling functions. FGF23 was assayed using ELISA kit in the presence or absence of 17β–estradiol in starved MLO-Y4 osteocytes. In these cells, a relationship between oxidative stress-induced apoptosis and up-regulation of active FGF23 levels due to MAP Kinases activation with involvement of the transcriptional factor (NF-kB) has been demonstrated. The active FGF23 increase can be due to up-regulation of its expression and post-transcriptional modifications. 17β–estradiol prevents the increase of FGF23 by inhibiting JNK and NF-kB activation, osteocyte apoptosis and by the down-regulation of osteoclastogenic factors, such as sclerostin. No alteration in the levels of dentin matrix protein 1, a FGF23 negative regulator, has been determined. The results of this study identify biological targets on which drugs and estrogen may act to control active FGF23 levels in oxidative stress-related bone and non-bone inflammatory diseases.

PanNENs are histopathologically classified as well-differentiated pancreatic neuroendocrine tumors (panNETs) or poorly differentiated pancreatic neuroendocrine carcinomas (panNECs) according to the 2010 World Health Organization (WHO) classification system. Pancreatic neuroendocrine tumors (pNETs) occur in 60–70% of multiple endocrine neoplasia (MEN1) patients; about 30% of pNETs undergo a malignant progression, manifesting local or distant metastases, which are one of the main causes of death in MEN1 patients.1, 2 Their molecular characteristics are still undefined. Moreover, despite the common genetic basis, their clinical phenotype is highly variable among patients, even in the presence of the same mutation, suggesting a possible role of other cofactors and/or epigenetic mechanisms in each individual tumorigenesis process.3-5 We analyzed, through next-generation sequencing (NGS), the specific miRNA expression signatures of normal pancreas, gastrinoma, and neuroendocrine pancreatic tumor in a MEN1 patient, whose histopathological examination (via duodenopancreatectomy biopsies) revealed diffuse microadenomatosis with eight macrotumors (>0.5 cm) in the pancreas and four gastrin-secreting tumors (three in the duodenum and one in the gallbladder) (Figure S1). NGS analysis of normal pancreas and MEN1 tumor samples identified the presence of tumor-subtype-specific miRNAs (see Figures 1 and 2). These data open to the possibility of using them for distinguishing different MEN1 gastro-entero-pancreatic neuroendocrine tumors (GEP-NETs). Principal component analysis (PCA) analysis allowed us to confirm that our normalized miRNA expression profiles are able to resolve samples according to their tissue. In particular, while biopsy replicates displayed a low amount of variance (Figure 1A and B), the first two principal components captured 87% of the overall variance and separated the control pancreas from MEN1 pNETs and gastrinomas (Figure 1B). To delve more deeply into these groups, we then proceeded to perform a robust statistical characterization of both subtype-specific and pan-cancer miRNAs. We first performed a group-wise study, in order to extract the miRNAs specifically up-/down-regulated in either pNETs or gastrinomas when compared to control pancreas (“pNET vs. CP” and “Gas vs. CP,” respectively). miRNAs with an adjusted p-value < 0.01 were considered differentially expressed (DE) between pairs of conditions (Tables 2 and 3), and are shown in Figure 1C–F. UpSet plot in Figure 2A shows the degree of intersection between up- (UP) and downregulated (DOWN) miRNAs in “pNET vs. CP” and “Gas vs. CP” contrasts. Our data demonstrate that most of the miRNAs analyzed are homogeneously split into pNET-specific, Gas-specific, and Common classes, while only <4% of them have a divergent behavior. The normalized expression values of miRNAs belonging to the four classes in panel A are shown as heatmaps (Figure 2B–E) and may contain a useful panel of subtype-specific biomarkers. We then focused on the class of miRNAs DE between MEN1 gastro-entero-pancreatic cancer tissues (pNETs and gastrinomas) and control pancreas. miRNAs with an adjusted p-value < 0.01 were considered DE between conditions (Table 4). We observed 17 downregulated and 26 upregulated miRNAs, whose log2 FoldChanges versus average expressions and volcano plots are shown in Figure 3A. These data were also shown in a heatmap containing the normalized expression value of differentially regulated miRNAs (Figure 3B). These miRNAs represent a miRNA signature specific for MEN1 gastro-entero-pancreatic tumors. Then, we applied a correlation network model to identify co-expressed miRNAs and study the impact of their regulation in the context of the network. The reconstructed network originated from MEN1 control and MEN1 tumor gastro-entero-pancreatic samples, and focused on DE miRNAs. The topology of this network evidenced two independent cliques (Figure 3C) corresponding to two clusters of anti-correlated miRNAs, and highlighted some hubs of marked connectivity. We hypothesized that hub miRNAs may act as proxies to help explaining the regulatory function played by the broader regulatory network. Therefore, we examined hub miRNAs from the two different cliques in a deeper analysis. First, to confirm deep sequencing results, we used qRT-PCR to assess the expression of seven hub miRNAs from the two different cliques (C1 clicque: miRNA-378-3p; miRNA-1468-5p; miR-625-5p; miR-625-3p; miR-215-5p. C2 clicque: miR-1301-3p; miR-212-5p). Importantly, the trend of the alteration in the expression was generally concordant (six out of seven) between the sequencing data and qRT-PCR (Figure 4A). Moreover, for most of these miRNAs, qRT-PCR results showed a good agreement with the expression levels found in the BON1 cell line (pancreatic carcinoid tumor), commonly used as a reference model for NETs. Next, we checked whether genes predicted to be targets of these hub miRNAs shared a common function. GSEA (Gene-set Enrichment Analysis) revealed that predicted gene targets (according to miRWalk 3.0) were associated to specific KEGG pathways (Figure 4B and C and Table 5) related to cancer establishment and signaling: noteworthy, the list of the top hits includes the “pancreatic cancer” pathway. Exerting their role as key determinants of gene expression, transcription factors and miRNAs are able to co-regulate the expression of targets in the form of feed-forward loops (FFLs) and feedback loops: feed-forward loop analysis evidenced interactions between hub miRNAs and the principal genes involved in the MEN1 gastro-entero-pancreatic neoplasia, as transcription factors (TF) and/or other mRNAs (Figure 4D and E).6 The analysis showed miRNAs “hubs” organized in FFLs with chromatin-remodeling genes (MEN1, ATRX, DAXX) involved in familial and sporadic cancer, as well as with classical oncogenes (RB, TP53) normally mutated in neuroendocrine carcinomas (NEC) (Figure 4D and E). Therefore, it is tempting to suggest that these epigenetic bistable molecular regulatory circuits7, 8 could influence the transition from normal neuroendocrine cells to either well-differentiated GEP-NET cancer or to a poorly differentiated NEC. In summary, by investigating global miRNA expression signatures in matched samples of normal pancreas, pNET, and gastrinoma from a MEN1 clinical case, here we highlighted new specific miRNA signatures and confirmed previously selected microRNAs involved in MEN1 gastro-entero-pancreatic tumors. These miRNAs have been shown to be organized in two gene regulatory networks with common and distinct functional attributes. While this work clearly represents a starting point, and investigation of a greater number of cases will be required to generalize the results here presented, these data make an interesting exploratory contribution to the knowledge of the mechanisms governing MEN1 tumorigenesis. The authors gratefully thank Prof. Stefano Colagrande (Department of Experimental and Clinical Biomedical Sciences, University of Florence). The tissue samples for the study were obtained from a female donor undergoing duodenopancreatic surgery in 2002. Samples were collected by the surgeon after verbal informed consent from the patient, who fully understood that the samples were to be analyzed anonymously for future research on MEN1 syndrome not necessarily related to her specific clinical case. All genetic and molecular analyses for this study were carried out anonymously. Researchers had only access to clinical data of the donor and pathologic characteristics of the samples. Tables containing raw and processed data from the bioinformatic pipeline (including miRNA counts and complete secondary analysis results) can be accessed at the following address: http://doi.org/10.17632/wfm74g24d7.1. The authors declare no conflict of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

CONTEXT:Hypophosphatasia (HPP) is a rare metabolic disorder caused by deficiency of alkaline phosphatase (ALP) enzyme activity, leading to defective mineralization, due to pathogenic variants of the ALPL gene, encoding the tissue nonspecific alkaline phosphatase (TNSALP) enzyme. Inheritance can be autosomal recessive or autosomal dominant. An abnormal ALPL genetic test enables accurate diagnosis, avoiding the administration of contraindicated antiresorptive drugs that, in patients with HPP, substantially increase the risk of atypical femur fractures (AFFs) and worsen the fracture healing process that is usually already compromised in these patients.OBJECTIVE:Performing ALPL genetic testing to identify rare variants in suspected adult patients with HPP. Comparing frequencies of ALPL common variants in individuals with biochemical and/or clinical signs suggestive of adult HPP and non-HPP controls, and among different clinical subgroups of patients with a clinical suspicion of adult HPP.METHODS:Patients with suspected adult HPP were retrospectively selected for the genetic testing of the ALPL gene. Patients included were from 3 main European Bone Units (Florence, Naples, and Geneva); 106 patients with biochemical and/or clinical signs suggestive of a mild form of HPP were included.RESULTS:Genetic testing led to the identification of a heterozygote rare variant in 2.8% of cases who were initially referred as suspected osteoporosis. The analysis of frequencies of ALPL common variants showed a high prevalence (30.8%) of homozygosity in subjects who developed an AFF, in association with normal serum total ALP activity.CONCLUSION:The results suggest homozygosity of common ALPL variants as a possible genetic mark of risk for these fractures.

Multiple endocrine neoplasia type 1 (MEN1) is a rare inherited tumor syndrome, characterized by the development of multiple neuroendocrine tumors (NETs) in a single patient. Major manifestations include primary hyperparathyroidism, gastro-entero-pancreatic neuroendocrine tumors, and pituitary adenomas. In addition to these main NETs, various combinations of more than 20 endocrine and non-endocrine tumors have been described in MEN1 patients. Despite advances in diagnostic techniques and treatment options, which are generally similar to those of sporadic tumors, patients with MEN1 have a poor life expectancy, and the need for targeted therapies is strongly felt. MEN1 is caused by germline heterozygous inactivating mutations of the MEN1 gene, which encodes menin, a tumor suppressor protein. The lack of a direct genotype–phenotype correlation does not permit the determination of the exact clinical course of the syndrome. One of the possible causes of this lack of association could be ascribed to epigenetic factors, including microRNAs (miRNAs), single-stranded non-coding small RNAs that negatively regulate post-transcriptional gene expression. Some miRNAs, and their deregulation, have been associated with MEN1 tumorigenesis. Recently, an extracellular class of miRNAs has also been identified (c-miRNAs); variations in their levels showed association with various human diseases, including tumors. The aim of this review is to provide a general overview on the involvement of miRNAs in MEN1 tumor development, to be used as possible targets for novel molecular therapies. The potential role of c-miRNAs as future non-invasive diagnostic and prognostic biomarkers of MEN1 will be discussed as well.

Osteoporosis, one of the leading causes of bone fractures, is characterized by low bone mass and structural deterioration of bone tissue, which are associated with a consequent increase in bone fragility and predisposition to fracture. Current screening tools are limited in estimating the proper assessment of fracture risk, highlighting the need to discover novel more suitable biomarkers. Genetic and environmental factors are both implicated in this disease. Increasing evidence suggests that epigenetics and, in particular, miRNAs, may represent a link between these factors and an increase of fracture risk. miRNAs are a class of small noncoding RNAs that negatively regulate gene expression. In the last decade, several miRNAs have been associated with the development of osteoporosis and bone fracture risk, opening up new possibilities in precision medicine. Recently, these molecules have been identified in several biological fluids, and the possible existence of a circulating miRNA (c-miRNA) signature years before the fracture occurrence is suggested. The aim of this review is to provide an overview of the c-miRNAs suggested as promising biomarkers for osteoporosis up until now, which could be helpful for early diagnosis and monitoring of treatment response, as well as fracture risk assessment, in osteoporotic patients.

Juvenile idiopathic arthritis (JIA) is the most common chronic arthritis of children and adolescents. Autoimmune mechanisms are suspected to have a central role in its development. Vitamin D is an immuno-modulator in a variety of conditions, including autoimmune diseases. Low levels of vitamin D have commonly been found in JIA patients, but the influence of this hormone insufficiency in JIA pathogenesis is still unclear. Vitamin D receptor (VDR) mediates a great majority of vitamin D biological activities; specific polymorphisms of the VDR gene have been associated with different biologic responses to vitamin D. In this study, we analysed clinical characteristics of a cohort of 103 Italian JIA patients. The distribution of VDR polymorphisms in affected patients versus healthy controls was evaluated, as well as if and how these polymorphic variants associate with different disease presentations (active disease vs non-active disease), different JIA subtypes, serum levels of 25-hydroxy-vitamin D and parathyroid hormone (PTH), and lumbar spine Z-score values (osteopenia vs normal bone mineral density). A great majority of our JIA patients (84.5%) showed a suboptimal vitamin D status, in many cases (84.1%) not solved by vitamin D supplementation. Vitamin D status resulted to be independent of VDR genotypes. ApaI genotypes showed a highly significant different distribution between JIA patients and unaffected controls, with both the TT genotype and the T allele significantly more frequent in patient group.

Osteoporosis is a multifactorial skeletal disease that is associated with both bone mass decline and microstructure damage. The fragility fractures-especially those affecting the femur-that embody the clinical manifestation of this pathology continue to be a great medical and socioeconomic challenge worldwide. The currently available diagnostic tools, such as dual energy X-ray absorptiometry, Fracture Risk Assessment Tool (FRAX) score, and bone turnover markers, show limited specificity and sensitivity; therefore, the identification of alternative approaches is necessary. As a result of their advantageous features, such as non-invasiveness, biofluid stability, and easy detection, circulating cell-free miRs are promising new potential biomarkers for the diagnosis of osteoporosis and low-traumatic fracture risk assessment. However, due to the absence of both standardized pre-analytical, analytical, and post-analytical protocols for their measurement and universally accepted guidelines for diagnostic use, their clinical utility is limited. The aim of this review was to record all the data currently available in the literature concerning the use of circulating microRNAs as both potential biomarkers for osteoporosis diagnosis and fragility fracture risk evaluation, and group them according to the experimental designs, in order to support a more conscious choice of miRs for future research in this field.

Osteoporosis (OP) is a multifactorial disorder in which environmental factors along with genetic variants and epigenetic mechanisms have been implicated. Long non-coding RNAs (lncRNAs) have recently emerged as important regulators of bone metabolism and OP aetiology. In this study, we analyzed the expression level and the genetic association of lncRNA GAS5 in OP patients compared to controls. Quantitative RT-PCR analysis of GAS5 was performed on the serum of 56 OP patients and 28 healthy individuals. OP subjects were divided into three groups of analysis: 29 with fragility fractures of lumbar spine (OP_VF), 14 with fragility fractures of femoral neck (OP_FF) and 13 without fractures (OP_WF). Genotyping of the rs145204276 insertion/deletion polymorphism has also been performed by Restriction fragment length polymorphism (RFLP) and direct sequencing analyses. Expression of circulating GAS5 is significantly increased in OP patients compared to controls (p < 0.01), with a statistically higher significance in fractured OP individuals vs. healthy subjects (p < 0.001). No statistically significant change was found in female OP patients; conversely, GAS5 is upregulated in the subgroup of fractured OP women sera (p < 0.01) and in all OP males (p < 0.05). Furthermore, a direct correlation between GAS5 expression level and parathyroid hormone (PTH) concentration was found in OP patients (r = 0.2930; p = 0.0389). Genetic analysis of rs145204276 revealed that the deletion allele was correlated with a higher expression of GAS5 in OP patients (0.22 ± 0.02 vs. 0.15 ± 0.01, ** p < 0.01). Our results suggest circulating GAS5 as a putative biomarker for the diagnosis and prognosis of OP and OP-related fractures.

Hyperparathyrodism-jaw tumor (HPT-JT) syndrome is an autosomal dominant disorder. Loss of function of the cell division cycle protein 73 homolog (CDC73) gene is responsible for the syndrome. This gene encodes an ubiquitously expressed 531 amino acid protein, parafibromin, that acts as a tumor suppressor. Loss of heterozygosity (LOH) of the CDC73 locus in many HPT-JT associated parathyroid tumors from patients with germline mutation is in accordance with Knudson's "two-hit" model for hereditary cancer. A 41-year-old man with mandible ossifying fibroma suffered from severe hypercalcemia due to parathyroid carcinoma (PC). Genetic analysis was performed to evaluate germinal and somatic CDC73 gene mutation as well as real-time qRT-PCR to quantify CDC73 mRNA, miR-155 and miR-664 expression levels. Immunohistochemistry and Western blotting (WB) assay were carried out to evaluate parafibromin protein expression. A novel heterozygous nonsense mutation, c.191-192 delT, was identified in the CDC73 gene. No CDC73 LOH was found in PC tissue, nor any differences in expression levels for CDC73 gene, miR-155 and miR-664 between PC and parathyroid adenoma control tissues. On the contrary, both immunohistochemistry and WB assay showed an approximate 90% reduction of parafibromin protein expression in PC. In conclusion, this study describes a novel germinal mutation, c.191-192 delT, in the CDC73 gene. Despite normal CDC73 gene expression, we found a significant decrease in parafibromin. We hypothesize that a gene silencing mechanism, possibly induced by microRNA, could play a role in determining somatic post-transcriptional inactivation of the wild type CDC73 allele.

miRNAs are small non-coding RNAs of about 18–25 nucleotides that negatively regulate gene expression at the post-transcriptional level. It was reported that a deregulation of their expression patterns correlates to the onset and progression of various diseases. Recently, these molecules have been identified in a great plethora of biological fluids, and have also been proposed as potential diagnostic and prognostic biomarkers. Actually, real time quantitative polymerase chain reaction is the most widely used approach for circulating miRNAs (c-miRNAs) expression profiling. Nevertheless, the debate on the choice of the most suitable endogenous reference genes for c-miRNAs expression levels normalization is still open. In this regard, numerous research groups are focusing their efforts upon identifying specific, highly stable, endogenous c-mRNAs. The aim of this review is to provide an overview on the reference genes currently used in the study of various pathologies, offering to researchers the opportunity to select the appropriate molecules for c-miRNA levels normalization, when their choosing is based upon literature data.

ContextHyperparathyroidism-jaw tumour (HPT-JT) syndrome is a rare autosomal dominant cause of familial hyperparathyroidism associated with ossifying fibromas (OF) of the maxillofacial bones and increased risk of parathyroid carcinoma, caused by inactivating germline mutation of the cell division cycle 73 (CDC73) gene.ObjectiveTo report the first Romanian family with HPT-JT and genetic screening of CDC73 gene.Subjects and MethodsMutational analysis of the CDC73 gene and genetic screening of the family of a proband with HPT-JT. Histological diagnosis of parathyroid tumors (WHO criteria) and immunohistochemistry (parafibromin) were performed.ResultsThree of the six screened family members had evidence of PHPT and surgically proven parathyroid tumours. Two of the three affected members had parathyroid carcinomas and one had two parathyroid adenomas. Genetic screening of CDC73 gene revealed that 4 of 6 patients showed a heterozygous germline deletion of one nucleotide: c.128-IVS1+1 delG. All the three affected patients, resulted to be carriers of the CDC73 mutation, but each one bearing a different CDC73 polymorphism.ConclusionsWe identified a new CDC73 germline mutation in a Romanian family of HPT-JT. Analysis of clinical phenotypes in the four mutated individuals confirmed the incomplete penetrance and the variable clinical expression of the disease.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Multiple Endocrine Neoplasia type 1 (MEN1) syndrome is a rare complex tumor-predisposing hereditary disorder, inherited in an autosomal dominant manner (OMIM 131100). MEN1 is characterized by tumors of the parathyroids, the neuroendocrine cells of the gastro-entero-pancreatic tract, and the anterior pituitary. The molecular mechanisms that control parathyroid tumorigenesis are still poorly understood. Here we studied the global microRNAs (miRNAs) expression profile in MEN1 parathyroid adenomas to understand the role of these regulatory factors in MEN1 parathyroid tumorigenesis. miRNA arrays containing 1890 human miRNAs were used to profile seven different MEN1 parathyroid adenomas (four presenting somatic loss of heterozygosity (LOH) at 11q13 and three still retaining one wild type copy of the MEN1 gene). Eight miRNAs in non-LOH MEN1 parathyroid adenomas and two miRNAs in LOH MEN1 parathyroid adenomas resulted to be differentially expressed, with a significant fold change, with respect to the control pool. Six microRNAs also resulted to be differentially expressed between LOH MEN1 parathyroid adenomas and non-LOH MEN1 parathyroid adenomas. Significantly differentially expressed miRNAs were all validated by SYBR green real-time quantitative RT-PCR. Pearson correlation coefficient indicated miR-4258, miR-664 and miR-1301 as the most significant miRNAs. In silico target-prediction and network analysis showed miR-664 and miR-1301 as organized in predicted GRNs with genes interested in parathyroid adenomas and carcinomas. In conclusion, our study identified three new miRNAs involved in the MEN1 parathyroid neoplasia, directly targeting genes associated with the development of different inheritable forms of parathyroid tumors. These identified miRNAs could be revealed as prognostic and diagnostic biomarkers for parathyroid tumors to improve the diagnosis of MEN1 neoplasia and other syndromes.

Adipose-derived stem/stromal cells (ASCs), together with adipocytes, vascular endothelial cells, and vascular smooth muscle cells, are contained in fat tissue.ASCs, like the human bone marrow stromal/stem cells (BM-SCs), can differentiate into several lineages (adipose cells, fibroblast, chondrocytes, osteoblasts, neuronal cells, endothelial cells, myocytes, and cardiomyocytes).They have also been shown to be immunoprivileged, and genetically stable in long-term cultures.Nevertheless, unlike the BMSCs, ASCs can be easily harvested in large amounts with minimal invasive procedures.The combination of these properties suggests that these cells may be a useful tool in tissue engineering and regenerative medicine.

To retrospectively evaluate the age of onset of MEN1-associated lesions in a group of affected children and adolescents and to compare the clinical features of our series with the evidence derived from the literature. The study population consisted of 22 Italian children and adolescents (age 6-31 years at the time of the inclusion in this study) all with a clinical and/or a genetic diagnosis of MEN1 performed before the age of 16 who have been followed-up regularly from 1998 to 2016 at the Regional Referral Center for Hereditary Endocrine Tumors. Clinical, biochemical, imaging and genetic data have been collected for each patient. Ten subjects (45.5%) have not yet presented any clinical/biochemical/radiological manifestation of MEN1 disease, whereas 12 patients (54.5%) developed at least one MEN1-associated endocrine manifestation. The second group of patients was significantly older than the first one. The most frequent manifestation was primary hyperparathyroidism (50%), followed by pituitary tumors (prolactinomas) (31.8%) and nonfunctioning pancreatic neuroendocrine tumors (9%). The earliest cases of primary hyperparathyroidism and prolactinoma were a 12-year-old girl and a 13-year-old boy, respectively. MEN1 disease seems to present with different features in children and adolescents from those in adults. Our study confirms the fundamental importance of screening for tumors in young MEN1 patients beginning in early childhood, in order to avoid diagnostic and therapeutic delays.