Functional characterization of a rare germline mutation in the gene encoding the cyclin-dependent kinase inhibitor p27Kip1 (CDKN1B) in a Spanish patient with multiple endocrine neoplasia-like phenotype

Abstract

ObjectiveThe aim of this study was to investigate the presence of germline mutations in theCDKN1Bgene that encodes the cyclin-dependent kinase (Cdk) inhibitor p27 in multiple endocrine neoplasia 1 (MEN1)-like Spanish index patients. TheCDKN1Bgene has recently been identified as a tumor susceptibility gene for MEN4, with six germline mutations reported so far in patients with a MEN-like phenotype but negative for MEN1 mutations.Design and methodsFifteen Spanish index cases with MEN-like symptoms were screened for mutations in theCDKN1Bgene and the mutant variant was studied functionally by transcription/translation assaysin vitroand in transiently transfected HeLa cells.ResultsWe report the identification of a heterozygous GAGA deletion in the 5′-UTR ofCDKN1B, NM_004064.3:c.-32_-29del, in a patient affected by gastric carcinoid tumor and hyperparathyroidism. This deletion falls inside the region that is responsible forCDKN1Btranscription and is predicted to destroy a secondary stem and loop structure that includes the GAGAGA element responsible for ribosome recruitment. Accordingly,in vitrostudies of coupled transcription/translation assays and transient transfection in HeLa cells showed that the GAGA deletion in theCDKN1B5′-UTR significantly impairs the transcription of downstream reporter luciferase (of ∼40–60%) and, possibly, the translation of the corresponding mRNA. This mutation was associated with a significant reduction in the amount ofCDKN1BmRNA in peripheral blood leukocytes from the patient, as demonstrated by quantitative real-time PCR.ConclusionsOur results confirm that germlineCDKN1Bmutations may predispose to a human MEN4 condition and add novel evidence that alteration in the transcription/translation rate ofCDKN1BmRNA might be the mechanism implicated in tumor susceptibility.