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      A Novel Mutation in the Upstream Open Reading Frame of the CDKN1B Gene Causes a MEN4 Phenotype

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          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.

          Author Summary

          Gene expression can be modulated at different steps on the way from DNA to protein including control of transcription, translation, and post-translational modifications. An abnormality in the regulation of mRNA and protein expression is a hallmark of many human diseases, including cancer. In some eukaryotic genes translation can be influenced by small DNA sequences termed upstream open reading frames (uORFs). These elements located upstream to the gene start codon may either negatively influence the ability of the translational machinery to reinitiate translation of the main protein or, much less frequently, stimulate protein translation by enabling the ribosomes to bypass cis-acting inhibitory elements. CDKN1B, which encodes the cell cycle inhibitor p27 KIP1, includes an uORF in its 5′UTR sequence. p27 KIP1 expression is often reduced in cancer, and germline mutations have been identified in CDKN1B in patients affected with a syndrome (MEN4) characterized by varying combinations of tumors in endocrine glands. Here we show that a small deletion in the uORF upstream to CDKN1B reduces translation reinitiation efficiency, leading to underexpression of p27 KIP1 and coinciding with tumorigenesis. This study describes a novel mechanism by which p27 KIP1 could be underexpressed in human tumors. In addition, our data provide a new insight to the unique pathogenic potential of uORFs in human diseases.

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          Author and article information

          [1 ]Department of Medicine, Endocrinology Unit, University of Padova, Padova, Italy
          [2 ]Department of Endocrinology, Barts and the London School of Medicine, Queen Mary University of London, London, United Kingdom
          [3 ]Familial Cancer Clinic and Oncoendocrinology, Veneto Institute of Oncology, IRCCS, Padova, Italy
          [4 ]Department of Endocrinology and Metabolism, Section of Endocrinology and Bone Metabolism, University of Pisa, Pisa, Italy
          [5 ]Institute of Pathology, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany
          [6 ]Laboratory of Translational Genomics, Centre for Integrative Biology, University of Trento, Trento, Italy
          [7 ]Department of Medicine, University of Padova, Padova, Italy
          University of Washington, United States of America
          Author notes

          The authors have declared that no competing interests exist.

          Conceived and designed the experiments: G Occhi, D Regazzo, V Sidarovich, A Quattrone. Performed the experiments: G Occhi, D Regazzo, F Boaretto, D Ciato, G Trivellin, E Pardi, NS Pellegata, S Bobisse, V Sidarovich. Analyzed the data: G Occhi, D Regazzo, F Boaretto, G Trivellin, NS Pellegata, V Sidarovich, A Quattrone. Contributed reagents/materials/analysis tools: S Ferasin, F Cetani, M Korbonits, NS Pellegata, G Opocher, F Mantero, C Scaroni, A Quattrone. Wrote the paper: G Occhi, A Quattrone. Contributed to improving the manuscript with comments and suggestions: D Regazzo, M Korbonits, G Trivellin, NS Pellegata, G Opocher.

          Role: Editor
          PLoS Genet
          PLoS Genet
          PLoS Genetics
          Public Library of Science (San Francisco, USA )
          March 2013
          March 2013
          21 March 2013
          : 9
          : 3
          23555276 3605397 PGENETICS-D-12-01826 10.1371/journal.pgen.1003350

          This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

          Pages: 11
          This work was supported by the Italian Ministry of University and Research (grant 2009YJTBAZ_003) and by the University of Padova (grant CPDA105053/10). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
          Research Article
          Gene Expression
          Protein Translation
          Genetic Mutation
          Mutation Types
          Human Genetics
          Autosomal Dominant
          Molecular Genetics
          Gene Regulation
          Genetics of Disease



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