Introduction: CDKN1B gene, which encodes a cyclin-dependent kinase (Cdk) inhibitor, regulates the progression throughout G1 to S cell cycle progression. CDKN1B loss-of-function germinal mutations cause the multiple endocrine neoplasia type 4 syndrome (MEN4). Objective: The aim of the study is the functional characterization of a new 4 bp deletion in CDKN1B 5′-UTR region, identified in an acromegalic patient. Materials and methods: We assessed a functional in vitro study, based on firefly luciferase reporter gene, on the deleted promoter of human (MCF-7), murine (AtT20/D16V-F2) and rat (GH3) cell lines. Total mRNA and proteins were extracted from peripheral blood of the patient and control subjects to analyze CDKN1B/p27Kip1 expression. In addition, we evaluated p27Kip1 expression in tissue sections of the patient’s GH-secreting pituitary adenoma by immunohistochemistry. Results: A novel heterozygous deletion in CDKN1B 5′-UTR region was identified in an acromegalic patient. The deletion extends from nucleotide −29 to −26 from the translation start site. Transcriptional activity of the deleted promoter is significantly decreased (~30–60% P<0.01). CDKN1B/p27Kip1 expression analysis of patient’s circulating leukocytes showed a significant reduction (~72%) in mRNA levels, while p27Kip1 protein levels are similar to WT controls. Immunohistochemistry shows a reduced p27Kip1 expression in the patient’s pituitary adenoma compared to the control. Conclusions: Our data show that the identified deletion causes a significant reduction in promoter transcriptional activity and in CDKN1B mRNA expression, indicating a putative role of the deletion in the patient’s disease. Further studies are needed order to understand whether the deletion could impact protein function.
Functional characterization of a new deletion in CDKN1B 5'-UTR region
TAGLIATI, Federico;GENTILIN, Erica;GAGLIANO, Teresa;BENFINI, Katiuscia;DEGLI UBERTI, Ettore;ZATELLI, Maria Chiara
2014
Abstract
Introduction: CDKN1B gene, which encodes a cyclin-dependent kinase (Cdk) inhibitor, regulates the progression throughout G1 to S cell cycle progression. CDKN1B loss-of-function germinal mutations cause the multiple endocrine neoplasia type 4 syndrome (MEN4). Objective: The aim of the study is the functional characterization of a new 4 bp deletion in CDKN1B 5′-UTR region, identified in an acromegalic patient. Materials and methods: We assessed a functional in vitro study, based on firefly luciferase reporter gene, on the deleted promoter of human (MCF-7), murine (AtT20/D16V-F2) and rat (GH3) cell lines. Total mRNA and proteins were extracted from peripheral blood of the patient and control subjects to analyze CDKN1B/p27Kip1 expression. In addition, we evaluated p27Kip1 expression in tissue sections of the patient’s GH-secreting pituitary adenoma by immunohistochemistry. Results: A novel heterozygous deletion in CDKN1B 5′-UTR region was identified in an acromegalic patient. The deletion extends from nucleotide −29 to −26 from the translation start site. Transcriptional activity of the deleted promoter is significantly decreased (~30–60% P<0.01). CDKN1B/p27Kip1 expression analysis of patient’s circulating leukocytes showed a significant reduction (~72%) in mRNA levels, while p27Kip1 protein levels are similar to WT controls. Immunohistochemistry shows a reduced p27Kip1 expression in the patient’s pituitary adenoma compared to the control. Conclusions: Our data show that the identified deletion causes a significant reduction in promoter transcriptional activity and in CDKN1B mRNA expression, indicating a putative role of the deletion in the patient’s disease. Further studies are needed order to understand whether the deletion could impact protein function.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
