Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry

Abstract

Methylation is one of the major epigenetic processes pivotal to our understanding of carcinogenesis. It is now widely accepted that there is a relationship between DNA methylation, chromatin structure, and human malignancies. DNA methylation is potentially an important clinical marker in cancer molecular diagnostics. Understanding epigenetic modifications in their biological context involves several aspects of DNA methylation analysis. These aspects include the de novo discovery of differentially methylated genes, the analysis of methylation patterns, and the determination of differences in the degree of methylation. Here we present a previously uncharacterized method for high-throughput DNA methylation analysis that utilizes MALDI-TOF mass spectrometry (MS) analysis of base-specifically cleaved amplification products. We use the IGF2͞H19 region to show that a single base-specific cleavage reaction is sufficient to discover methylation sites and to determine methylation ratios within a selected target region. A combination of cleavage reactions enables the complete evaluation of all relevant aspects of DNA methylation, with most CpGs represented in multiple reactions. We successfully applied this technology under high-throughput conditions to quantitatively assess methylation differences between normal and neoplastic lung cancer tissue samples from 48 patients in 47 genes and demonstrate that the quantitative methylation results allow accurate classification of samples according to their histopathology. lung cancer ͉ MALDI-TOF MS ͉ epigenetics

CpG island methylator phenotype-low (CIMP-low) colorectal cancer shows not only few methylated CIMP-high-specific CpG islands, but also low-level methylation at individual loci

“…Thus, quantitative assessment of DNA methylation is very important. 24,25,27,39,40 Our findings provide additional supporting evidence for molecular features of CIMP-low tumors different from CIMP-high and CIMP-0 tumors. CIMP-low tumors generally appear to methylate at multiple CIMP-specific CpG islands at low levels, whereas CIMP-0 tumors tend to show more loci with no evidence of methylation and CIMP-high tumors tend to show more loci with high-level methylation.…”

DNA methylation is associated with expression level changes of galectin gene in mantle wound healing process of pearl oyster, Pinctada fucata

“…After the in vitro transcription, 0.06 mL RNase A was added to cleave the in vitro transcript. The mixture was diluted with ddH 2 O, dispensed onto silicon chips preloaded with matrix (SpectroCHIP; SEQUENOM, Inc.), and the mass spectra data were collected by the MassARRAY mass spectrometer (Bruker-SEQUENOM) [26].…”

Tissue-specific differentially methylated regions of the human VASA gene are potentially associated with maturation arrest phenotype in the testis

“…Histology also revealed MA in testicular biopsy results obtained from 17 patients, who were defined as the MA group in this study. Although all MA patients lacked late spermatogenesis when examined histologically, an examination of ejaculate revealed that some had few spermatozoa in the ejaculate (MA4, 8,27), as shown in Tables 1 and 2. The mean ages of the control and MA groups (±s.d.)…”

“…27 This system analyzed base-specific cleaved amplification products. In this study, we used the MassARRAY Compact system (Sequenom).…”