title: Functional characterization of a CDKN1B mutation in a Sardinian kindred with multiple endocrine neoplasia type 4 (MEN4).
abstract: 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.
authors.affiliation: E Pardi, Department of Clinical and Experimental Medicine, University of Pisa, Endocrine Unit 2, University Hospital of Pisa, Pisa, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Pardi
authors.affiliation: S Mariotti, Endocrinology Unit, Department of Medical Sciences "M Aresu", University of Cagliari, Cagliari, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Mariotti
authors.affiliation: N Pellegata, Institute of Pathology, Helmholtz Zentrum Muenchen, Neuherberg, Germany.
authors.forename: Natalia S
authors.initials: NS
authors.lastname: Pellegata
authors.affiliation: K Benfini, Institute of Pathology, Helmholtz Zentrum Muenchen, Neuherberg, Germany.
authors.forename: Katiuscia
authors.initials: K
authors.lastname: Benfini
authors.affiliation: S Borsari, Department of Clinical and Experimental Medicine, University of Pisa, Endocrine Unit 2, University Hospital of Pisa, Pisa, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Borsari
authors.affiliation: F Saponaro, Department of Clinical and Experimental Medicine, University of Pisa, Endocrine Unit 2, University Hospital of Pisa, Pisa, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Saponaro
authors.affiliation: L Torregrossa, Surgical, Medical and Molecular Pathology and Critical Area, University of Pisa, Pisa, Italy.
authors.forename: Liborio
authors.initials: L
authors.lastname: Torregrossa
authors.affiliation: A Cappai, Endocrinology Unit, Department of Medical Sciences "M Aresu", University of Cagliari, Cagliari, Italy.
authors.forename: Antonello
authors.initials: A
authors.lastname: Cappai
authors.affiliation: C Satta, Endocrinology Unit, Department of Medical Sciences "M Aresu", University of Cagliari, Cagliari, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Satta
authors.affiliation: M Mastinu, Endocrinology Unit, Department of Medical Sciences "M Aresu", University of Cagliari, Cagliari, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Mastinu
authors.affiliation: C Marcocci, Department of Clinical and Experimental Medicine, University of Pisa, Endocrine Unit 2, University Hospital of Pisa, Pisa, Italy.
authors.forename: Claudio
authors.initials: C
authors.lastname: Marcocci
authors.affiliation: F Cetani, Department of Clinical and Experimental Medicine, University of Pisa, Endocrine Unit 2, University Hospital of Pisa, Pisa, Italy cetani@endoc.med.unipi.it.
authors.forename: Filomena
authors.initials: F
authors.lastname: Cetani