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Selective Loss of MEG3 Expression and Intergenic Differentially Methylated Region Hypermethylation in the MEG3/DLK1 Locus in Human Clinically Nonfunctioning Pituitary Adenomas

Neuroendocrine Unit (R.G., D.L.B., Y.Zho, Y.Zhou, X.Z., A.K.), Neuropathology Unit (R.G., E.T.H.-W.), and Division of Neurosurgery (B.S.), Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114; and Section on Endocrinology and Genetics and Pediatric Endocrinology Training Program (C.A.S.), Developmental Endocrinology Branch, National Institute of Child Health and Human Development, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Anne Klibanski, M.D., Neuroendocrine Unit, Massachusetts General Hospital, 55 Fruit Street, BUL457, Boston, Massachusetts 02114. E-mail: aklibanski{at}partners.org.

Context: MEG3 is an imprinted gene encoding a novel noncoding RNA that suppresses tumor cell growth. Although highly expressed in the normal human pituitary, it is unknown which of the normal pituitary cell types and pituitary tumors express MEG3.

Objectives: Our objectives were 1) to investigate cell-type- and tumor-type-specific expression of MEG3 in the human pituitary and 2) to investigate whether methylation in the intergenic differentially methylated region (IG-DMR) at the DLK1/MEG3 locus is involved in the loss of MEG3 expression in tumors.

Design and Methods: RT-PCR, quantitative RT-PCR, Northern blot, and a combination of in situ hybridization and immunofluorescence were used to determine the cell-type- and tumor-type-specific MEG3 expression. Bisulfite treatment and PCR sequencing of genomic DNA were used to measure the CpG methylation status in the normal and tumor tissues. Five normal human pituitaries and 17 clinically nonfunctioning, 11 GH-secreting, seven prolactin-secreting, and six ACTH-secreting pituitary adenomas were used.

Results: All normal human pituitary cell types express MEG3. However, loss of MEG3 expression occurs only in nonfunctioning pituitary adenomas of a gonadotroph origin. All other pituitary tumor phenotypes examined express MEG3. Hypermethylation of the IG-DMR at the DLK1/MEG3 locus is present in nonfunctioning pituitary adenomas.

Conclusions: MEG3 is the first human gene identified expressed in multiple normal human pituitary cell types with loss of expression specifically restricted to clinically nonfunctioning pituitary adenomas. The IG-DMR hypermethylation may be an additional mechanism for MEG3 gene silencing in such tumors.