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.

OBJECTIVE: The aim of this study was to investigate the presence of germline mutations in the CDKN1B gene that encodes the cyclin-dependent kinase (Cdk) inhibitor p27 in multiple endocrine neoplasia 1 (MEN1)-like Spanish index patients. The CDKN1B gene 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 METHODS: Fifteen Spanish index cases with MEN-like symptoms were screened for mutations in the CDKN1B gene and the mutant variant was studied functionally by transcription/translation assays in vitro and in transiently transfected HeLa cells.

RESULTS: We report the identification of a heterozygous GAGA deletion in the 5'-UTR of CDKN1B, 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 for CDKN1B transcription and is predicted to destroy a secondary stem and loop structure that includes the GAGAGA element responsible for ribosome recruitment. Accordingly, in vitro studies of coupled transcription/translation assays and transient transfection in HeLa cells showed that the GAGA deletion in the CDKN1B 5'-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 of CDKN1B mRNA in peripheral blood leukocytes from the patient, as demonstrated by quantitative real-time PCR.

CONCLUSIONS: Our results confirm that germline CDKN1B mutations may predispose to a human MEN4 condition and add novel evidence that alteration in the transcription/translation rate of CDKN1B mRNA might be the mechanism implicated in tumor susceptibility.