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

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 0 -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 0 -UTR significantly impairs the transcription of downstream reporter luciferase (of w40-60%) 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.

A heterozygous frameshift mutation in exon 1 of CDKN1B gene in a patient affected by MEN4 syndrome

“…Both functioning (GH or ACTH) and non-functioning tumors have been observed (4,5,6,7,8). The present case showed an increase in the values of prolactin without relevant morphological evidence of a lesion at the pituitary level.…”

“…A mutated transcript can determine a truncated protein or an altered protein mainly localized in the cytoplasm rather than in the nucleus with a lower stability and a diminished CDK2 binding property (6). During MEN4 tumorigenesis, the principal mechanism responsible for the reduced expression of p27 seems to be the posttranslational ubiquitin-proteasome degradation of the protein (1,7,14) and only in rare cases the reduced p27 expression is ascribable to CDKN1B somatic mutations or tissutal somatic CDKN1B LOH (5, 6). The p27 protein mainly localized to the cytoplasm is rapidly degraded through ubiquitylation by the KPC ubiquitin ligase and proteasome-mediated degradation (15,16,17) in tumors, which is different from the normal conditions.…”

“…PHPT has a high penetrance in MEN4 and, similar to MEN1, is the first diagnosed endocrinopathy in most cases, but with an age at onset more than two decades later than in MEN1 (mean 56 years in MEN4 vs 20-25 years in MEN1) (1,4,5,6,7,8). In about half of the MEN4 patients, only one gland was affected and responsible for PHPT, without genotype/phenotype relationships ever described regarding the severity and the number of the involved parathyroids (1,4,5,6,7,8). The follow-up of these patients is often lacking, and no information on the persistence or recurrence of PHPT is usually referred.…”

“…Heterozygote inactivating mutations of the CDKN1B gene have been evidenced in !2% of patients screened for the presence of a MEN1 phenotype not harboring MEN1 gene mutations (1,4,5,6,7,8,9,10), with a total of eight different germline mutations of the CDKN1B gene having been published so far (Table 1). This new syndrome, that in humans presents with an autosomal dominant pattern of inheritance, named MEN4 (4) (OMIM 610755, gene locus 600788 on chromosome 12p13.1), to distinguish it from MEN1, MEN2A (OMIM 171400, gene locus 164761 on chromosome 10q11.21), and MEN2B (formerly MEN3) (OMIM 162300, gene locus 164761 on chromosome 10q11.21), exhibits MEN1-like clinical manifestations but not MEN2-associated tumors in all the reported conditions (Table 1) (1, 4,5,6,7,8,9,10). In this paper, we report a case of a Caucasian female patient with a long clinical history suggestive of a MEN1 syndrome, but with a negative MEN1 genetic test, in which a novel germline heterozygote frameshift mutation of the CDKN1B gene has been identified.…”

MEN1 intragenic deletions may represent the most prevalent somatic event in sporadic primary hyperparathyroidism

“…This entity clinically closer to pHPT is characterised by parathyroid involvement and, less typically, by pituitary adenomas and other endocrine features (7). Until now, six germline CDKN1B mutations have been found in patients with a MEN1-like phenotype but were negative for MEN1 mutations (8). At variance with the wellknown genetic alterations underneath familial pHPT, the molecular alterations underlying the sporadic forms still remain poorly understood.…”