Thompson RF, Suzuki M, Lau KW et al.A pipeline for the quantitative analysis of CG dinucleotide methylation using mass spectrometry. Bioinformatics 25:2164-2170

Department of Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Bioinformatics (Impact Factor: 4.98). 07/2009; 25(17):2164-70. DOI: 10.1093/bioinformatics/btp382
Source: PubMed


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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Available from: Reid F Thompson, Nov 11, 2014
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    • "A total of 500 ng genomic DNA from each sample was bisulfite-treated using an EZ-96 DNA Methylation Kit (D5003; Zymo Research, Orange, CA). The quality of the bisulfite conversion was evaluated using MassARRAY analytical tools based on R language (Thompson et al., 2009), using the MassARRAY raw data for the peak value of the fragment containing at least one non-CG cytosine. Peak values of fragments with C or T were compared using the bisulfite conversion percent. "
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    ABSTRACT: BACKGROUND Animal models of neural tube defects (NTDs) have indicated roles for the Fzd3 gene and the planar cell polarity signaling pathway in convergent extension. We investigated the involvement of FZD3 in genetic and epigenetic mechanisms associated with human NTDs, especially spina bifida. We explored the effects of variants spanning the FZD3 gene in NTDs and examined the role of aberrant methylation of the FZD3 promoter on gene expression in brain tissue in spina bifida.METHODS Six FZD3 single nucleotide polymorphisms were genotyped using a MassARRAY system in tissue from 165 NTD fetuses and 152 controls. DNA methylation aberrations in the FZD3 promoter region were detected using a MassARRAY EpiTYPER (17 CpG units from −500 to −2400 bp from the transcription start site) in brain tissue from 77 spina bifida and 74 control fetuses.RESULTSNone of the six single nucleotide polymorphisms evaluated were significantly associated with spina bifida, but the mean methylation level was significantly higher in spina bifida samples (13.70%) compared with control samples (10.91%) (p = 0.001). In terms of specific sites, DNA methylation levels were significantly higher in the spina bifida samples at 14 of the 17 CpG units, which mostly included in R2 region. FZD3 mRNA expression was negatively correlated with methylation of the FZD3 promoter region, especially the R2 region (R = 0.970; p = 0.001) in HeLa cells.CONCLUSION The results of this study suggest that DNA methylation plays an important role in FZD3 gene expression regulation and may be associated with an increased risk of spina bifida. Birth Defects Research (Part A), 2014. © 2014 Wiley Periodicals, Inc.
    Birth Defects Research Part A Clinical and Molecular Teratology 08/2014; 103(1). DOI:10.1002/bdra.23285 · 2.09 Impact Factor
    • "Quality control for the EpiTyper assay included double measurement of amplicon 2 containing six CpG sites in all samples, which showed high concordance. Testing of bisulfite conversion rates was applied using the bioconductor package MassArray (Thompson et al., 2009). Samples showing a conversion rate o80% were excluded from analysis. "

    41st Annual Meeting of the Arbeitsgemeinschaft-Dermatologische-Forschung; 03/2014
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    • "This combination provides a highly accurate, sensitive and high-throughput method for the quantitative analysis of DNA methylation. The EpiTYPER software provides convenient solutions for data analysis and export, however,the Sequenom EpiTYPER has a 5% technical error rate [33]. Although the robustness of this approach for confirming the accuracy of Sequenom methylation analysis results has been demonstrated, we used two alternate methods,pyrosequence and BSP,to test the methylation analysis results of NKX2-5 and GATA4. "
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    ABSTRACT: NKX2-5, GATA4 and HAND1 are essential for heart development, however, little is known regarding their epigenetic regulation in the pathogenesis of tetralogy of fallot (TOF). Methylation levels were measured in three regions of NKX2-5 (M1: -1596 bp ~ -1374 bp, M2: -159 bp ~ 217 bp and M3: 1058 bp ~ 1524 bp), one region of GATA4 (M: -392 bp ~ 107 bp) and three regions of HAND1 (M1: -887 bp ~ -414 bp, M2: -436 bp ~ 2 bp and M3: 37 bp ~ 398 bp) using the Sequenom MassARRAY platform. QRT-PCR was used to analyze NKX2-5 and HAND1 mRNA levels in the right ventricular myocardium of TOF patients. TOF patients had a significantly higher NKX2-5_M3 median methylation level than controls (41.65% vs. 22.18%; p = 0.0074; interquartile range [IQR]: 30.46%--53.35%, N = 30 and 20.07%--24.31%, N = 5; respectively). The HAND1_M1 median methylation level was also significantly higher in TOF patients than controls (30.05% vs. 17.54%; p = 0.0054; IQR: 20.77%--40.89%, N = 30 and IQR: 14.69%--20.64%; N = 6; respectively). The methylation statuses of NKX2-5_M1, NKX2-5_M2, GATA4_M, HAND1_M2 or HAND1_M3 were not significantly different in TOF patients compared to controls. The methylation values for NKX2-5_M3 were negatively correlated with mRNA levels (r = - 0.463, p = 0.010, N = 30) and there was a significant association between HAND1_M1 methylation status and mRNA levels (r = - 0.524, p = 0.003, N = 30) in TOF patients. Aberrant methylation statuses of the NKX2-5 gene body and HAND1 promoter regions are associated with the regulation of gene transcription in TOF patients and may play an important role in the pathogenesis of TOF.
    BMC Medical Genomics 11/2013; 6(1):46. DOI:10.1186/1755-8794-6-46 · 2.87 Impact Factor
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