A pipeline for the quantitative analysis of CG dinucleotide methylation using mass spectrometry.

Department of Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Bioinformatics (Impact Factor: 4.62). 07/2009; 25(17):2164-70. DOI: 10.1093/bioinformatics/btp382
Source: PubMed

ABSTRACT MOTIVATION: DNA cytosine methylation is an important epigenetic regulator, critical for mammalian development and the control of gene expression. Numerous techniques using either restriction enzyme or affinity-based approaches have been developed to interrogate cytosine methylation status genome-wide, however these assays must be validated by a more quantitative approach, such as MALDI-TOF mass spectrometry of bisulphite-converted DNA (commercialized as Sequenom's EpiTYPER assay using the MassArray system). Here, we present an R package ('MassArray') that assists in assay design and uses the standard Sequenom output file as the input to a pipeline of analyses not available as part of the commercial software. The tools in this package include bisulphite conversion efficiency calculation, sequence polymorphism flagging and visualization tools that combine multiple experimental replicates and create tracks for genome browser viewing.

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    ABSTRACT: Single nucleotide polymorphisms in GSDMB and ORMDL3 are strongly associated with childhood asthma but the molecular alterations contributing to disease remain unknown. We investigated the effects of asthma-associated SNPs on DNA methylation and mRNA-levels of GSDMB and ORMDL3. Genetic association between GSDMB/ORMDL3 and physician-diagnosed childhood asthma was confirmed in the Swedish birth-cohort BAMSE. CpG-site SNPs (rs7216389 and rs4065275) showed differences in DNA methylation depending on carrier status of the risk alleles, and were significantly associated with methylation levels in two CpG sites in the 5'UTR of ORMDL3. In the Swedish Search study, we found significant differences in DNA methylation between asthmatics and controls in five CpG sites; after adjusting for lymphocyte and neutrophil cell counts, three remained significant: one in IKZF3 (cg16293631) and two in the CpG island of ORMDL3 (cg02305874 and cg16638648). Also, cg16293631 and cg02305874 correlated with mRNA levels of ORMDL3. The association of methylation and asthma was independent of the genotype in rs7216389, rs4065275 and rs12603332. Both SNPs and CpG sites showed significant associations with ORMDL3 mRNA levels. SNPs influenced expression independently of methylation and the residual association between methylation and expression was not mediated by these SNPs. We found a differentially methylated region in the CpG island shore of ORMDL3 with six CpG sites less methylated in CD8(+)T-cells. In summary, this study supports that there are differences in DNA methylation at this locus between asthmatics and controls; and both SNPs and CpG sites are independently associated with ORMDL3 expression.
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