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

Abstract

The CDKN1B gene encodes the cyclin-dependent kinase inhibitor p27KIP1, an atypical tumor suppressor playing a key role in cell cycle regulation, cell proliferation, and differentiation. Impaired p27KIP1 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 p27KIP1 expression. Our findings demonstrate that, in addition to the previously described mechanisms leading to reduced p27KIP1 activity, such as degradation via the ubiquitin/proteasome pathway or non-covalent sequestration, p27KIP1 activity can also be modulated by an uORF and mutations affecting uORF could change p27KIP1 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.

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

“…Following this original description, other patients presenting with a MEN1 phenotype, but no germline mutations in the MEN1 gene, were described (1,4,10). In the evaluated cases, CDKN1B gene mutations generate a decrease in its encoded product, through various mechanisms, encompassing the synthesis of a truncated protein and/or the reduction of either protein expression (4,5,6,8). One of the identified mutations consists of a nucleotide variation at a stop codon associated with the formation of a protein longer than normal, mostly localized in the cytoplasm rather than in the nucleus, with a low stability and a reduced CDK2 binding affinity (1).…”

“…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.…”

15 YEARS OF PARAGANGLIOMA: The association of pituitary adenomas and phaeochromocytomas or paragangliomas

“…A pathogenic truncating mutation in the human orthologue CDKN1B was identified in a 48-year-old woman with a personal history of acromegaly and primary hyperparathyroidism and a family history of renal Printed in Great Britain angiomyolipoma in a confirmed mutation carrier (Pellegata et al 2006). Subsequently, a number of cases of both functional (Georgitsi et al 2007, Agarwal et al 2009, Tichomirowa et al 2012, Occhi et al 2013, Sambugaro et al 2015 and non-functional (Molatore et al 2010) PAs have been reported in patients with germline mutations in CDKN1B, although they account for only a minority of MEN1 mutation negative patients (Ozawa et al 2007, Igreja et al 2009). Mutations in other cyclin-dependent kinase inhibitors have also been linked to MEN.…”

A giant? Think of genetics: growth hormone-producing adenomas in the young are almost always the result of genetic defects

“…The defect led to a reduction in CDKN1B mRNA levels, altered secondary mRNA structure, and reduced transcriptional activity of the gene in laboratory studies [14]. The report by Sambugaro and Di Ruvo et al follows the identification of another 4-bp deletion (c.-456_-453delCCTT) in a highly conserved regulatory region of the 5 0 UTR of the CDKN1B gene in a patient with a pituitary adenoma and a well-differentiated pancreatic neoplasm [15].…”