MEN1 is a rare form of cancer that pre-disposes those affected to a wide range of endocrine specific malignancies and abnormalities, including development of tumours in the pancreas, anterior pituitary and parathyroid glands. Familial MEN1 is inherited in an autosomal dominant manner. A large family of 3000 individuals affected by a MEN1 mutation was identified in Tasmania. Family members carry a splice site mutation (C>G) inherited from the founding parents. Interestingly, disease severity and outcome varies substantially between patients carrying the same mutation. The reason for this is as yet unclear.
Given that methylation marks are known to alter gene expression and to be important in cancer etiology we hypothesise that variable methylation of the promoter region may contribute to observed changes in severity.
Forty-five abnormal FFPE parathyroid samples were available for analysis from ten patients in the Tasman 1 family, together with matched peripheral blood samples. Three normal blood controls (2 female, 1 male) along with an additional two phenocopy patient samples (blood and parathyroid) were analysed. Methylation at specific sites of the MEN1 promoter was examined by bisulphite sequencing. Primers were designed to amplify two areas of interest (island A and region B) in the promoter, encompassing a total of 39 CpG dinucleotides.
Variable levels of promoter methylation were observed across islands in abnormal parathyroid tissue. Low methylation was observed within island A, consistent with the published literature. Detailed examination of the promoter revealed a second area with high CG content, (region B), further upstream of the transcription initiation site. This region is previously unexplored and we report hypermethylation in abnormal MEN1 parathyroid when compared with phenocopy parathyroid tissue. Immunohistochemical staining for MEN1 was undertaken to examine protein expression. Methylation status and protein expression were examined in conjunction with clinical indicators of disease.