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K Revill, ISTM, Keele University, Stoke-on-Trent, United Kingdom
K Dudley, ISTM, Keele University, Stoke-on-Trent, United Kingdom
A McNicol, Molecular and Cellular Pathology, The University of Queensland, Brisbane, Australia
R Clayton, Dept of Medicine, University of Keele, North Staffordshire Hospital Centre, stoke, United Kingdom
W Farrell, ISTM, Keele University, Stoke-on-Trent, United Kingdom

Correspondence: William Farrell, Email: w.e.farrell{at}keele.ac.uk

Abstract

The imprinted gene, Neuronatin (NNAT), is one of the most abundant transcripts in the pituitary and is thought to be involved in the development and maturation of this gland. In a recent whole-genome approach, exploiting a pituitary tumour cell line, we identified hypermethylation associated loss of NNAT. In this report we determined the expression pattern of NNAT in individual cell types of the normal gland and within each of the different pituitary adenoma subtypes. In addition, we determined associations between expression and CpG island methylation and used colony forming efficiency assays (CFE) to gain further insight into the tumour-suppressor function of this gene. Immunohistochemical (IHC) co-localisation studies of normal pituitaries showed that each of the hormone secreting cells (Growth hormone-GH, Prolactin-PRL, Adrenocorticotroph hormone-ACTH, Follicle stimulating hormone-FSH and Thyroid stimulating hormone-TSH) expressed NNAT. However, 33 of 47 adenomas comprising, 11 somatotrophinomas, 10 prolactinomas, 12 corticotrophinomas and 14 non-functioning tumours, irrespective of subtype failed to express either NNAT transcript or protein as determined by RT-qPCR and IHC respectively. In normal pituitaries and adenomas that expressed NNAT the promoter-associated CpG island showed characteristics of an imprinted gene where ~50% of molecules were densely methylated. However, in the majority of adenomas that showed loss or significantly reduced expression of NNAT relative to normal pituitaries the gene-associated CpG island showed significantly increased methylation. Induced expression of NNAT in transfected AtT20 cells significantly reduced CFE. Collectively these findings point to an important role for NNAT in the pituitary and perhaps tumour development in this gland.