Quantitative, genome-wide analysis of the DNA methylome in sporadic pituitary adenomas

Abstract

DNA methylation is one of the several epigenetic modifications that together with genetic aberrations are hallmarks of tumorigenesis including those emanating from the pituitary gland. In this study, we examined DNA methylation across 27 578 CpG sites spanning more than 14 000 genes in the major pituitary adenoma subtypes. Genome-wide changes were first determined in a discovery cohort comprising non-functioning (NF), growth hormone (GH), prolactin (PRL)-secreting and corticotroph (CT) adenoma relative to post-mortem pituitaries. Using stringent cut-off criteria, we validated increased methylation by pyrosequencing in 12 of 16 (75%) genes. Overall, these criteria identified 40 genes in NF, 21 in GH, six in PRL and two in CT that were differentially methylated relative to controls. In a larger independent cohort of adenomas, for genes in which hypermethylation had been validated, different frequencies of hypermethylation were apparent, where the KIAA1822 (HHIPL1) and TFAP2E genes were hypermethylated in 12 of 13 NF adenomas whereas the COL1A2 gene showed an increase in two of 13 adenomas. For genes showing differential methylation across and between adenoma subtypes, pyrosequencing confirmed these findings. In three of 12 genes investigated, an inverse relationship between methylation and transcript expression was observed where increased methylation of EML2, RHOD and HOXB1 is associated with significantly reduced transcript expression. This study provides the first genome-wide survey of adenoma, subtype-specific epigenomic changes and will prove useful for identification of biomarkers that perhaps predict or characterise growth patterns. The functional characterisation of identified genes will also provide insight of tumour aetiology and identification of new therapeutic targets.

Epidrug mediated re-expression of miRNA targeting the HMGA transcripts in pituitary cells

“…[3,4,2,5] The relative contributions of these aberrations, their order of appearance, and the way in which their effects are manifest are largely unknown, however, specific aberration, be they genetic or epigenetic, are either common or particular to an adenoma subtype(s). [6,5,7] Moreover, the demarcation between genetic and epigenetic aberrations are perhaps less precise than initial findings suggested. In this context, we now know that the GNAS1 gene (the gsp oncogene), is activated or inappropriately expressed, either through mutation or relaxation of imprinting in growth hormone secreting pituitary adenomas.…”

“…PCR amplifications and pyrosequencing was performed as previously described by us. [6] Chromatin Immunoprecipitation (ChIP) analysis for histone modifications:…”

936 Quantitative genome-wide methylation analysis of high-grade non-muscle invasive bladder cancer

“…Indeed, similar observations with respect to differences in the frequencies of methylation between high-and low-grade bladder tumors were first suggested by Ibragimova et al 47 Similar subtype and/or grade-associated differences have been reported in other tumor types including, pituitary, breast, and colon cancer subtypes. 37,48,49 In our analysis of NMIBC it remains unclear whether the increase in frequency and/or mean levels of methylation in the more aggressive tumors represents a more rapid accumulation of epigenetic changes during tumor progression, or reflects distinct epigenetic pathways of tumor development and outgrowth. 50,51 Our findings may therefore reflect either of the described scenarios in the more aggressive (high-grade) tumors and suggests that these tumors are either consequent to progression from low-intermediategrade tumors, or are the progeny of aberrations in distinct epigenetic pathways within these NMIBC subtypes.…”

“…34,35,36 These analyses also showed that for the majority of regions investigated, methylation extended to include contiguous promoter-associated CpG sites. On the basis of previous reports from our own and other groups, 37,38 we employed stringent criteria (b-value differences 0.4) to identify differentially methylated genes across multiple CpG sites; such criteria are more consistently associated with bona fide changes in methylation, and are more likely to show associations with gene expression. 37,39,40,41 The analysis of the discovery cohort of high-grade NMIBC identified 1,057 CpGs, across 256 gene-promoter-associated CpG islands.…”

“…On the basis of previous reports from our own and other groups, 37,38 we employed stringent criteria (b-value differences 0.4) to identify differentially methylated genes across multiple CpG sites; such criteria are more consistently associated with bona fide changes in methylation, and are more likely to show associations with gene expression. 37,39,40,41 The analysis of the discovery cohort of high-grade NMIBC identified 1,057 CpGs, across 256 gene-promoter-associated CpG islands. Cluster analysis and heat map display of these regions revealed extensive and frequent differential methylation in the tumors relative to normal bladder controls.…”