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Purpose: Multiple endocrine neoplasia type 4 (MEN4) is a rare multiglandular endocrine neoplasia syndrome, associated with a wide tumor spectrum but hallmarked by primary hyperparathyroidism, which represents the most common clinical feature, followed by pituitary (functional and non-functional) adenomas, and neuroendocrine tumors. MEN4 clinically overlaps MEN type 1 (MEN1) but differs from it for milder clinical features and an older patient's age at onset. The underlying mutated gene, CDKN1B, encodes the cell cycle regulator p27, implicated in cellular proliferation, motility and apoptosis. Given the paucity of MEN4 cases described in the literature, possible genotype-phenotype correlations have not been thoroughly assessed, and specific clinical recommendations are lacking. The present review provides an extensive overview of molecular genetics and clinical features of MEN4, with the aim of contributing to delineate peculiar strategies for clinical management, screening and follow-up of the last and least known MEN syndrome., Methods: A literature search was performed through online databases like MEDLINE and Scopus., Conclusions: MEN4 is much less common that MEN1, tend to present later in life with a more indolent course, although involving the same primary organs as MEN1. As a consequence, MEN4 patients might need specific diagnostic and therapeutic approaches and a different strategy for screening and follow-up. Further studies are needed to assess the real oncological risk of MEN4 carriers, and to establish a standardized screening protocol. Furthermore, a deeper understanding of molecular genetics of MEN4 is needed in order to explore p27 as a novel therapeutic target., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Pheochromocytomas (PCCs) are tumors arising from neural crest-derived chromaffin cells. There are currently few animal models of PCC that recapitulate the key features of human tumors. Because such models may be useful for investigations of molecular pathomechanisms and development of novel therapeutic interventions, we characterized a spontaneous animal model (multiple endocrine neoplasia [MENX] rats) that develops endogenous PCCs with complete penetrance. Urine was longitudinally collected from wild-type (wt) and MENX-affected (mutant) rats and outputs of catecholamines and their O-methylated metabolites determined by mass spectrometry. Adrenal catecholamine contents, cellular ultrastructure, and expression of phenylethanolamine N-methyltransferase, which converts norepinephrine to epinephrine, were also determined in wt and mutant rats. Blood pressure was longitudinally measured and end-organ pathology assessed. Compared with wt rats, mutant animals showed age-dependent increases in urinary outputs of norepinephrine (P = .0079) and normetanephrine (P = .0014) that correlated in time with development of tumor nodules, increases in blood pressure, and development of hypertension-related end-organ pathology. Development of tumor nodules, which lacked expression of N-methyltransferase, occurred on a background of adrenal medullary morphological and biochemical changes occurring as early as 1 month of age and involving increased adrenal medullary concentrations of dense cored vesicles, tissue contents of both norepinephrine and epinephrine, and urinary outputs of metanephrine, the metabolite of epinephrine. Taken together, MENX-affected rats share several biochemical and pathophysiological features with PCC patients. This model thus provides a suitable platform to study the pathogenesis of PCC for preclinical translational studies aimed at the development of novel therapies for aggressive forms of human tumors.

Multiple endocrine neoplasia (MEN) syndromes are autosomal dominant diseases with high penetrance characterized by proliferative lesions (usually hyperplasia or adenoma) arising in at least two endocrine tissues. Four different MEN syndromes have been so far identified: MEN type 1 (MEN1), MEN2A (also referred to as MEN2), MEN2B (or MEN3) and MEN4, which have slightly varying tumor spectra and are caused by mutations in different genes. MEN1 associates with loss-of-function mutations in the MEN1 gene encoding the tumor suppressor menin. The MEN2A and MEN2B syndromes are due to activating mutations in the proto-oncogene RET (Rearranged in Transfection) and are characterized by different phenotypic features of the affected patients. MEN4 was the most recent addition to the family of the MEN syndromes. It was discovered less than 10 years ago thanks to studies of a rat strain that spontaneously develops multiple endocrine tumors (named MENX). These studies identified an inactivating mutation in the Cdkn1b gene, encoding the putative tumor suppressor p27, as the causative mutation of the rat syndrome. Subsequently, germline mutations in the human ortholog CDKN1B were also found in a subset of patients with a MEN-like phenotype and this led to the identification of MEN4. Small animal models have been instrumental in understanding important biochemical, physiological and pathological processes of cancer onset and spread in intact living organisms. Moreover, they have provided us with insight into gene function(s) and molecular mechanisms of disease progression. We here review the currently available animal models of MEN syndromes and their impact on the elucidation of the pathophysiology of these diseases, with a special focus on the rat MENX syndrome that we have been characterizing., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Ghrelin, the natural ligand of the growth hormone secretagogue receptor type 1a (GHS-R1a), is mainly secreted from the stomach and regulates food intake and energy homeostasis. p27 regulates cell cycle progression in many cell types. Here, we report that rats affected by the multiple endocrine neoplasia syndrome MENX, caused by a p27 mutation, develop pancreatic islet hyperplasia containing elevated numbers of ghrelin-producing ε-cells. The metabolic phenotype of MENX-affected rats featured high endogenous acylated and unacylated plasma ghrelin levels. Supporting increased ghrelin action, MENX rats show increased food intake, enhanced body fat mass, and elevated plasma levels of triglycerides and cholesterol. Ghrelin effect on food intake was confirmed by treating MENX rats with a GHS-R1a antagonist. At 7.5 months, MENX-affected rats show decreased mRNA levels of hypothalamic GHS-R1a, neuropeptide Y (NPY), and agouti-related protein (AgRP), suggesting that prolonged hyperghrelinemia may lead to decreased ghrelin efficacy. In line with ghrelin's proposed role in glucose metabolism, we find decreased glucose-stimulated insulin secretion in MENX rats, while insulin sensitivity is improved. In summary, we provide a novel nontransgenic rat model with high endogenous ghrelin plasma levels and, interestingly, improved glucose tolerance. This model might aid in identifying new therapeutic approaches for obesity and obesity-related diseases, including type 2 diabetes., (© 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Unlabelled: We evaluated (18)F-LMI1195 (1-(3-bromo-4-(3-(18)F-fluoro-propoxy)benzyl)guanidine), a metaiodobenzylguanidine (MIBG) analog, for the detection of pheochromocytoma in a preclinical in vivo model of endogenous neuroendocrine tumors (multiple endocrine neoplasia [MENX])., Methods: Adrenal uptake kinetics of (18)F-LMI1195 were evaluated in healthy Wistar rats (n = 6) by dynamic PET imaging. Distribution of (18)F-LMI1195 was evaluated in tumor-bearing MENX mut/mut rats (n = 10) and control MENX wild-type rats (n = 4) by biodistribution studies and PET imaging. Biodistribution of (18)F-LMI1195 was compared with (123)I-MIBG in MENX mut/mut rats (n = 6) and correlated with histological tumor volume and norepinephrine transporter (NET) expression. Uptake specificity was evaluated by in vivo inhibition of the NET by desipramine (n = 6). Intraadrenal distribution of (18)F-LMI1195 was evaluated by autoradiography., Results: (18)F-LMI1195 showed rapid tracer accumulation in adrenal glands 1 min after tracer injection. Adrenal glands of MENX mut/mut animals showed significantly higher standardized uptake value than MENX wild-type controls (maximum SUV, 10.3 ± 2.3 vs. 6.1 ± 0.9, P < 0.01). Adrenal uptake in MENX mut/mut rats could be inhibited by desipramine, shown by biodistribution studies (0.06 ± 0.01 vs. 0.16 ± 0.05 percentage injected dose, P < 0.01), PET imaging (maximum SUV, 3.8 ± 0.8 vs. 10.3 ± 2.3, P < 0.01), and autoradiography. Adrenal uptake of (18)F-LMI1195 correlated with (123)I-MIBG uptake (r = 0.91), histological tumor volume (r = 0.68), and NET expression (r = 0.50). (18)F-LMI1195 showed an overall favorable distribution for tumor imaging., Conclusion: (18)F-LMI1195 shows high and specific accumulation in pheochromocytomas. Its favorable biodistribution makes it a promising PET tracer for tumor imaging. Further studies are warranted to evaluate its clinical value in oncologic indications.

Multiple endocrine neoplasias (MEN) are autosomal dominant disorders characterized by the occurrence of tumors in at least two endocrine glands. Until recently, two MEN syndromes were known, i.e. the MEN type 1 (MEN1) and type 2 (MEN2), which are caused by germline mutations in the MEN1 and RET genes, respectively. These two syndromes are characterized by a different tumor spectrum. A few years ago we described a variant of the MEN syndromes, which spontaneously developed in a rat colony and was named MENX. Affected animals consistently develop multiple endocrine tumors, with a spectrum that shares features with both MEN1 and MEN2 human syndromes. Genetic studies identified a germline mutation in the Cdkn1b gene, encoding the p27 cell cycle inhibitor, as the causative mutation for MENX. Capitalizing on these findings, germline mutations in the human homologue, CDKN1B, were searched for and identified in patients with multiple endocrine tumors. As a consequence of this discovery, a novel human MEN syndrome, named MEN4, was recognized, which is caused by heterozygous mutations in p27. These studies identified Cdkn1b/CDKN1B as a novel tumor susceptibility gene for multiple endocrine tumors in both rats and humans. Here we review the characteristics of the MENX and MEN4 syndromes and we briefly address the main function of p27 and how it is affected by MENX- or MEN4-associated mutations.

Aims: The aim of this study is to evaluate the pathological features, serum hormone levels and ex vivo cultures of pituitary adenomas that occur in rats affected by MENX syndrome. MENX is multiple endocrine neoplasia syndrome caused by a germline mutation in the cell cycle inhibitor p27. Characterization of MENX adenomas is a prerequisite to exploit this animal model for molecular and translational studies of pituitary adenomas., Methods: We investigated MENX pituitary adenomas with immunohistochemistry, double immunofluorescence, electron microscopy, reverse transcription polymerase chain reaction (RT-PCR), measurement of serum hormone levels and ex vivo cultures., Results: Adenomas in MENX rats belong to the gonadotroph lineage. They start from 4 months of age as multiple neoplastic nodules and progress to become large lesions that efface the gland. Adenomas are composed of chromophobic cells predominantly expressing the glycoprotein alpha-subunit (αGSU). They show mitotic activity and high Ki67 labelling. A few neoplastic cells co-express gonadotropins and the transcription factor steroidogenic factor 1, together with growth hormone or prolactin and Pit-1, suggesting that they are not fully committed to one cell lineage. Ex vivo cultures show features similar to the primary tumour., Conclusions: Our results suggest that p27 function is critical to regulate gonadotroph cells growth. The MENX syndrome represents a unique model to elucidate the physiological and molecular mechanisms mediating the pathogenesis of gonadotroph adenomas., (© 2012 The Authors. Neuropathology and Applied Neurobiology © 2012 British Neuropathological Society.)

The CDKN1B gene encodes the cyclin-dependent kinase inhibitor p27(KIP1), an atypical tumor suppressor playing a key role in cell cycle regulation, cell proliferation, and differentiation. Impaired p27(KIP1) expression and/or localization are often observed in tumor cells, further confirming its central role in regulating the cell cycle. Recently, germline mutations in CDKN1B have been associated with the inherited multiple endocrine neoplasia syndrome type 4, an autosomal dominant syndrome characterized by varying combinations of tumors affecting at least two endocrine organs. In this study we identified a 4-bp deletion in a highly conserved regulatory upstream ORF (uORF) in the 5'UTR of the CDKN1B gene in a patient with a pituitary adenoma and a well-differentiated pancreatic neoplasm. This deletion causes the shift of the uORF termination codon with the consequent lengthening of the uORF-encoded peptide and the drastic shortening of the intercistronic space. Our data on the immunohistochemical analysis of the patient's pancreatic lesion, functional studies based on dual-luciferase assays, site-directed mutagenesis, and on polysome profiling show a negative influence of this deletion on the translation reinitiation at the CDKN1B starting site, with a consequent reduction in p27(KIP1) expression. Our findings demonstrate that, in addition to the previously described mechanisms leading to reduced p27(KIP1) activity, such as degradation via the ubiquitin/proteasome pathway or non-covalent sequestration, p27(KIP1) activity can also be modulated by an uORF and mutations affecting uORF could change p27(KIP1) expression. This study adds the CDKN1B gene to the short list of genes for which mutations that either create, delete, or severely modify their regulatory uORFs have been associated with human diseases., Competing Interests: The authors have declared that no competing interests exist.

A few years ago a novel multiple endocrine neoplasia syndrome, named multiple endocrine neoplasia type 4 (MEN4), was discovered thanks to studies conducted on a MEN syndrome in the rat (named MENX). The rat and the human syndromes are both caused by germline mutations in the Cdkn1b/CDKN1B gene, respectively. This gene encodes p27Kip1, a putative tumor suppressor which binds to and inhibits cyclin/cyclin-dependent kinase complexes, thereby preventing cell cycle progression. MEN4 patients carry heterozygous mutations at various residues of p27Kip1 and present with endocrine lesions mainly belonging to a MEN1-like spectrum: their most common phenotypic features are parathyroid and pituitary adenomas. Recently, germline mutations in p27kip1 were also identified in patients with a sporadic parathyroid disease presentation. In vitro functional analysis of several CDKN1B sequence changes identified in MEN4 patients detected impaired activity of the encoded p27Kip1 variant proteins (e.g. reduced expression, mislocalization or poor binding to interaction partners), thereby highlighting the characteristics of the protein which are critical for tumor suppression. Although the number of MEN4 patients is low, the discovery of this syndrome has demonstrated a novel role for CDKN1B as a tumor susceptibility gene for neuroendocrine tumors. Here, we review the clinical characteristics of the MEN4 syndrome and the molecular phenotype of the associated p27Kip1 mutations., (Copyright © 2013 S. Karger AG, Basel.)

Multiple endocrine neoplasias (MEN) are a group of hereditary disorders characterized by tumors arising in more than one neuroendocrine tissue. There are two major forms which can occur in humans, MEN type 1 (MEN1) and MEN type 2 (MEN2). These syndromes are transmitted as autosomal dominant traits with high penetrance and have a different tumor spectrum. MEN1 and MEN2 are caused by germline mutations in the MEN1 and RET genes, respectively. Recently, a variant of the MEN syndromes was discovered in a rat colony and was named MENX since affected animals develop tumors with a spectrum that shares features with both MEN1 and MEN2 human syndromes. Extensive genetic studies identified a germline mutation in the Cdkn1b gene, encoding the p27 cell cycle inhibitor, as the causative mutation for MENX. Capitalizing on these findings, heterozygous germline mutations in the human homologue, CDKN1B, were searched for and identified in patients with multiple endocrine tumors. As a consequence of this discovery, a novel human MEN syndrome, named MEN4, was recognized which is caused by mutations in p27. Altogether these studies identified Cdkn1b/CDKN1B as a novel tumor susceptibility gene for multiple endocrine tumors in both rats and humans. Here I review the phenotypic features and the genetics of the MENX rat syndrome. I briefly address the main functions of p27 and how they are affected by the MENX-associated mutation. Finally, I present examples of how this animal model might be exploited as a translational platform for preclinical studies of pituitary adenomas., (Copyright © 2012. Published by Elsevier Masson SAS.)

Multiple endocrine neoplasias are autosomal dominant disorders characterized by the occurrence of tumors in at least two endocrine glands. Two MEN syndromes have long been known and are well characterized: the MEN type 1 (MEN1) and type 2 (MEN2). These syndromes are caused by germline mutations in the MEN1 and RET genes, respectively, and have a different tumor spectrum. Recently, a variant of the MEN syndromes arose spontaneously in a rat colony and was named MENX. Affected animals consistently develop multiple endocrine tumors, with a spectrum that shares features with both MEN1 and MEN2 human syndromes. Genetic studies identified a germline mutation in the Cdkn1b gene, encoding the p27 cell cycle inhibitor, as the causative mutation for MENX. Capitalizing on these findings, heterozygous germline mutations in the human homologue, CDKN1B, were searched for and identified in patients with multiple endocrine tumors. As a consequence of this discovery, a novel human MEN syndrome, named MEN4, was recognized, which is caused by mutations in p27. Altogether, these studies identified Cdkn1b/CDKN1B as a novel tumor susceptibility gene for multiple endocrine tumors in both rats and humans. Here we review the characteristics of the MENX and MEN4 syndromes and we briefly address the main function of p27 and how they are affected by MENX/4-associated mutations.

Multiple endocrine neoplasias (MEN) are autosomal dominant disorders characterized by the occurrence of tumors in at least two endocrine glands. Two types of MEN syndromes have long been known: MEN type 1 (MEN1) and MEN type 2 (MEN2), associated with a different spectrum of affected organs. MEN1 and MEN2 are caused by germline mutations in the MEN1 tumor suppressor gene and the RET proto-oncogene, respectively. Lately, a new type of MEN was identified (named MEN4) which is due to mutations in the CDKN1B gene, encoding for p27kip1 (p27), a cyclin-dependent kinase (Cdk) inhibitor that regulates the transition of cells from G1 to S phase. p27 is a non-canonical tumor suppressor since it is usually not somatically mutated in human cancers but it is often downregulated by post-translational mechanisms. The discovery of MEN4 has defined a new role for CDKN1B as a tumor susceptibility gene for multiple endocrine tumors. To date, six germline CDKN1B mutations have been found in patients with a MEN1-like phenotype but negative for MEN1 mutations. Due to the limited number of patients so far identified, the phenotypic features of MEN4 are not clearly defined. Here, we review the clinical and molecular characteristics of the MEN4 syndrome and summarize the main functions of p27 to better comprehend how their alteration can predispose to neuroendocrine tumors., (Copyright © 2010 S. Karger AG, Basel.)

Multiple endocrine neoplasia (MEN) syndromes are characterized by tumors involving two or more endocrine glands. Two MEN syndromes have long been known: MEN1 and MEN2,caused by germline mutations in MEN1 or RET, respectively. Recently, mutations in CDKN1B,encoding the cyclin-dependent kinase (Cdk) inhibitor p27, were identified in patients having a MEN1-like phenotype but no MEN1 gene mutations. Currently, the molecular mechanisms mediating the role of p27 in tumor predisposition are ill defined. We here report a novel germline missense variant in CDKN1B (c.678C>T, p.P69L) found in a patient with multiple endocrine tumors. We previously reported a nonsense p27 mutation (c.692G>A, p.W76X) in two patients with MEN1-like phenotype. Functional assays were used to characterize p27P69L and p27W76X in vitro. We show that p27P69L is expressed at reduced level and is impaired in both binding toCdk2 and inhibiting cell growth. p27W76X, which is mislocalized to the cytoplasm, can no longer efficiently bind Cyclins-Cdks, nor inhibit cell growth or induce apoptosis. In the patient’s tumor tissues, p27P69L associates with reduced/absent p27 expression and in one tumor with loss-of heterozygosity.Our results extend previous findings of CDKN1B mutations in patients with MEN1-related states and support the hypothesis of a tumor suppressor role for p27 in neuroendocrine cells., (©2010 Wiley-Liss, Inc.)

Pheochromocytomas are rare neoplasias of neural crest origin arising from chromaffin cells of the adrenal medulla and sympathetic ganglia (extra-adrenal pheochromocytoma). Pheochromocytoma that develop in rats homozygous for a loss-of-function mutation in p27Kip1 (MENX syndrome) show a clear progression from hyperplasia to tumor, offering the possibility to gain insight into tumor pathobiology. We compared the gene-expression signatures of both adrenomedullary hyperplasia and pheochromocytoma with normal rat adrenal medulla. Hyperplasia and tumor show very similar transcriptome profiles, indicating early determination of the tumorigenic signature. Overrepresentation of developmentally regulated neural genes was a feature of the rat lesions. Quantitative RT-PCR validated the up-regulation of 11 genes, including some involved in neural development: Cdkn2a, Cdkn2c, Neurod1, Gal, Bmp7, and Phox2a. Overexpression of these genes precedes histological changes in affected adrenal glands. Their presence at early stages of tumorigenesis indicates they are not acquired during progression and may be a result of the lack of functional p27Kip1. Adrenal and extra-adrenal pheochromocytoma development clearly follows diverged molecular pathways in MENX rats. To correlate these findings to human pheochromocytoma, we studied nine genes overexpressed in the rat lesions in 46 sporadic and familial human pheochromocytomas. The expression of GAL, DGKH, BMP7, PHOX2A, L1CAM, TCTE1, EBF3, SOX4, and HASH1 was up-regulated, although with different frequencies. Immunohistochemical staining detected high L1CAM expression selectively in 27 human pheochromocytomas but not in 140 nonchromaffin neuroendocrine tumors. These studies reveal clues to the molecular pathways involved in rat and human pheochromocytoma and identify previously unexplored biomarkers for clinical use.

Background: p27Kip1 (p27) is an important negative regulator of the cell cycle and a putative tumor suppressor. The finding that a spontaneous germline frameshift mutation in Cdkn1b (encoding p27) causes the MENX multiple endocrine neoplasia syndrome in the rat provided the first evidence that Cdkn1b is a tumor susceptibility gene for endocrine tumors. Noteworthy, germline p27 mutations were also identified in human patients presenting with endocrine tumors. At present, it is not clear which features of p27 are crucial for this tissue-specific tumor predisposition in both rats and humans. It was shown that the MENX-associated Cdkn1b mutation causes reduced expression of the encoded protein, but the molecular mechanisms are unknown. To better understand the role of p27 in tumor predisposition and to characterize the MENX animal model at the molecular level, a prerequisite for future preclinical studies, we set out to assess the functional properties of the MENX-associated p27 mutant protein (named p27fs177) in vitro and in vivo., Results: In vitro, p27fs177 retains some properties of the wild-type p27 (p27wt) protein: it localizes to the nucleus; it interacts with cyclin-dependent kinases and, to lower extent, with cyclins. In contrast to p27wt, p27fs177 is highly unstable and rapidly degraded in every phase of the cell-cycle, including quiescence. It is in part degraded by Skp2-dependent proteasomal proteolysis, similarly to p27wt. Photobleaching studies showed reduced motility of p27fs177 in the nucleus compared to p27wt, suggesting that in this compartment p27fs177 is part of a multi-protein complex, likely together with the degradation machinery. Studies of primary rat newborn fibroblasts (RNF) established from normal and MENX-affected littermates confirmed the rapid degradation of p27fs177 in vivo which can be rescued by Bortezomib (proteasome inhibitor drug). Overexpression of the negative regulators microRNA-221/222 plays no role in regulating the amount of p27fs177 in RNFs and rat tissues., Conclusion: Our findings show that reduced p27 levels, not newly acquired properties, trigger tumor formation in rats, similarly to what has been observed in mice. The molecular characteristics of p27fs177 establish MENX as a useful preclinical model to evaluate compounds that inhibit p27 degradation for their efficacy against endocrine tumors.

In the past 3 years new insight into the etiopathogenesis of hereditary endocrine tumors has emerged from studies conducted on MENX, a rat multiple endocrine neoplasia (MEN) syndrome. MENX spontaneously developed in a rat colony and was discovered by serendipity when these animals underwent complete necropsy, as they were found to consistently develop multiple endocrine tumors with a spectrum similar to both MEN type 1 (MEN1) and MEN2 human syndromes. Genetic studies identified a germline mutation in the Cdkn1b gene, encoding the p27 cell cycle inhibitor, as the causative mutation for the MENX syndrome. Capitalizing on these findings, we and others identified heterozygous germline mutations in the human homologue, CDKN1B, in patients with multiple endocrine tumors. As a consequence of these observations a novel human MEN syndrome, named MEN4, was recognized which is caused by mutations in p27. Altogether these studies identified Cdkn1b/CDKN1B as a novel tumor susceptibility gene for multiple endocrine tumors in both rats and humans. In this chapter we present the MENX syndrome and its phenotype, and we compare it to the human MEN syndromes; we discuss the current state of knowledge regarding the genes associated to inherited MEN, with a particular focus on CDKN1B; we present recent clinical and basic findings about the MEN4 syndrome and the functional characterization of the CDKN1B mutations identified. These findings are placed in the broader context of how p27 dysregulation might affect neuroendocrine cell function and trigger tumorigenesis., (Copyright 2010 Elsevier B.V. All rights reserved.)

MENX is a recessive multiple endocrine neoplasia-like syndrome in the rat. The tumor spectrum in MENX overlaps those of human multiple endocrine neoplasia (MEN) types 1 and 2. We mapped the MenX locus to the distal part of rat chromosome 4, excluding the homologs of the genes responsible for the MEN syndromes (RET and MEN1) and syndromes with an endocrine tumor component (VHL and NF1). We report the fine mapping of the disease locus and the identification of a homozygous frameshift mutation in Cdkn1b, encoding the cyclin-dependent kinase inhibitor p27(Kip1). As a consequence of the mutation, MENX-affected rats show dramatic reduction in p27(Kip1) protein. We have identified a germ-line nonsense mutation in the human CDKN1B gene in a MEN1 mutation-negative patient presenting with pituitary and parathyroid tumors. Expanded pedigree analysis shows that the mutation is associated with the development of an MEN1-like phenotype in multiple generations. Our findings demonstrate that germ-line mutations in p27(Kip1) can predispose to the development of multiple endocrine tumors in both rats and humans.

Multiple endocrine neoplasia-like syndrome (MENX) is a hereditary cancer syndrome in the rat characterized by inborn cataract and multiple tumors affecting the neuroendocrine system developed within the first year of life. The spectrum of affected organs is intermediate between MEN type 1 (MEN1) and MEN type 2 (MEN2) syndromes in human, but, in contrast to them, MENX is inherited in a recessive fashion. Here we report the mapping of the MENX locus to rat Chromosome (Chr) 4 by a genome-wide linkage analysis. This analysis was done in 41 animals obtained from a (Wistar/Nhg x SDwe) x SDwe interstrain backcross, where SDwe (Sprague-Dawley white eye) indicates the affected animals. The MENX disease locus was ultimately mapped to a approximately 22-cM interval on Chr 4 that includes the rat homolog of the human RET proto-oncogene. As activating point mutations of RET are known to be responsible for MEN2 in human, we analyzed several markers located in the proximity of Ret for linkage to the disease phenotype. Our data exclude Ret involvement in MENX and establish that a second gene, playing a role in endocrine tumor formation, lies within the distal part of rat Chr 4. Although heritable human endocrine tumors are quite rare, sporadic tumors of MEN-affected tissues occur at a much higher frequency, and their pathogenesis is poorly understood. The identification of the MENX gene should contribute to our understanding of the genetic mechanisms of neuroendocrine tissue tumorigenesis and may assist in developing new and more appropriate therapeutic strategies for these diseases.