Genetic alterations frequently are involved in the development of a pituitary adenoma in young age. We here characterize the functional role of a deletion in CDKN1B 5'-UTR region (c.-29_-26delAGAG) identified in an acromegalic patient that developed a growth hormone in pituitary adenoma during childhood. Our results show that the identified novel heterozygous deletion in the CDKN1B 5'-UTR region associates with a reduction in CDKN1B mRNA levels, a predicted altered secondary mRNA structure, and a reduced CDKN1B 5'-UTR transcriptional activity in vitro. The patient displayed loss of heterozygosity in the same CDKN1B 5'-UTR region at tissue level and the 5'UTR region containing the deleted sequence encompasses a GRE. These findings indicate that the identification of functional alterations of newly discovered genetic derangements need to be fully characterized and always correlated with the clinical manifestations.

Multiple endocrine neoplasia type 1 (MEN1) syndrome is an autosomal dominant disease, characterized by parathyroid adenomas, endocrine gastroenteropancreatic tumors and pituitary adenomas, due to inactivating mutations of the MEN1 gene (chromosome 11q13). MEN1 mutations are mainly represented by nonsense, deletions/insertions, splice site or missense mutations that can be detected by direct sequencing of genomic DNA. However, MEN1 patients with large heterozygous deletions may escape classical genetic screening and may be misidentified as phenocopies, thereby hindering proper clinical surveillance. We employed a real-time polymerase chain reaction application, the TaqMan copy number variation assay, to evaluate a family in which we failed to identify an MEN1 mutation by direct sequencing, despite a clear clinical diagnosis of MEN1 syndrome. Using the TaqMan copy number variation assay we identified a large deletion of the MEN1 gene involving exons 1 and 2, in three affected family members, but not in the other nine family members that were to date clinically unaffected. The same genetic alteration was not found in a group of ten unaffected subjects, without family history of endocrine tumors. The MEN1 deletion was further confirmed by multiplex ligation-dependent probe amplification, which showed the deletion extended from exon 1 to exon 3. This new approach allowed us to correctly genetically diagnose three clinical MEN1 patients that were previously considered as MEN1 phenocopies. More importantly, we excluded the presence of genetic alterations in the unaffected family members. These results underline the importance of using a variety of available biotechnology approaches when pursuing a genetic diagnosis in a clinically suggestive setting of inherited endocrine cancer.

CH extract displays a glucocorticoid-like activity, by means of a direct binding to the glucocorticoid receptor.

Introduction: CDKN1B gene, which encodes a cyclin-dependent kinase (Cdk) inhibitor, regulates the progression throughout G1 to S cell cycle progression. CDKN1B loss-of-function germinal mutations cause the multiple endocrine neoplasia type 4 syndrome (MEN4). Objective: The aim of the study is the functional characterization of a new 4 bp deletion in CDKN1B 5′-UTR region, identified in an acromegalic patient. Materials and methods: We assessed a functional in vitro study, based on firefly luciferase reporter gene, on the deleted promoter of human (MCF-7), murine (AtT20/D16V-F2) and rat (GH3) cell lines. Total mRNA and proteins were extracted from peripheral blood of the patient and control subjects to analyze CDKN1B/p27Kip1 expression. In addition, we evaluated p27Kip1 expression in tissue sections of the patient’s GH-secreting pituitary adenoma by immunohistochemistry. Results: A novel heterozygous deletion in CDKN1B 5′-UTR region was identified in an acromegalic patient. The deletion extends from nucleotide −29 to −26 from the translation start site. Transcriptional activity of the deleted promoter is significantly decreased (~30–60% P<0.01). CDKN1B/p27Kip1 expression analysis of patient’s circulating leukocytes showed a significant reduction (~72%) in mRNA levels, while p27Kip1 protein levels are similar to WT controls. Immunohistochemistry shows a reduced p27Kip1 expression in the patient’s pituitary adenoma compared to the control. Conclusions: Our data show that the identified deletion causes a significant reduction in promoter transcriptional activity and in CDKN1B mRNA expression, indicating a putative role of the deletion in the patient’s disease. Further studies are needed order to understand whether the deletion could impact protein function