Recommendations for the design and analysis of epigenome-wide association studies

Michels, Karin B.; Binder, Alexandra M.; Dedeurwaerder, Sarah; Epstein, Charles B.; Greally, John M.; Gut, Marta; Houseman, E. Andrés; Izzi, Benedetta; Kelsey, Karl T.; Meissner, Alexander; Milosavljevic, Aleksandar; Siegmund, Kimberly D.; Bock, Christoph; Irizarry, Rafael A.

doi:10.1038/nmeth.2632

Cited by 352 publications

(322 citation statements)

References 51 publications

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“…19 We have focused on a purified population of cells from which preexisting functional evidence suggests are relevant to the pathogenesis of disease. Despite this, CD4+ T-cells are heterogeneous and, therefore, we utilized a surrogate variable analysis to capture unwanted heterogeneity and included these effects as covariates in the linear modeling.…”

Section: Discussionmentioning

confidence: 99%

Epigenome-wide association study reveals longitudinally stable DNA methylation differences in CD4+ T cells from children with IgE-mediated food allergy

et al. 2014

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Section: Discussionmentioning

confidence: 99%

Epigenome-wide association study reveals longitudinally stable DNA methylation differences in CD4+ T cells from children with IgE-mediated food allergy

et al. 2014

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“…Thus, exposure to inflammation during fetal life may link growth restriction with subsequent development of type 2 diabetes [17]. While genes displaying differential methylation associated with IUGR have been identified in peripheral blood lymphocytes (PBLs) [9], the cell heterogeneity of the samples may have introduced experimental artefacts that could alter findings [18].…”

Section: Introductionmentioning

confidence: 99%

DNA hypermethylation of CD3+ T cells from cord blood of infants exposed to intrauterine growth restriction

et al. 2016

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Aims/hypothesis Intrauterine growth restriction (IUGR) is associated with increased susceptibility to obesity, metabolic syndrome and type 2 diabetes. Although the mechanisms underlying the developmental origins of metabolic disease are poorly understood, evidence suggests that epigenomic alterations play a critical role. We sought to identify changes in DNA methylation patterns that are associated with IUGR in CD3 + T cells purified from umbilical cord blood obtained from male newborns who were appropriate for gestational age (AGA) or who had been exposed to IUGR. Methods CD3 + T cells were isolated from cord blood obtained from IUGR and AGA infants. The genome-wide methylation profile in eight AGA and seven IUGR samples was determined using the HELP tagging assay. Validation analysis using targeted bisulfite sequencing and bisulfite massARRAY was performed on the original cohort as well as biological replicates consisting of two AGA and four IUGR infants. The Segway algorithm was used to identify methylation changes within regulatory regions of the genome. Results A global shift towards hypermethylation in IUGR was seen compared with AGA (89.8% of 4,425 differentially methylated loci), targeted to regulatory regions of the genome, specifically promoters and enhancers. Pathway analysis identified dysregulation of pathways involved in metabolic disease (type 2 diabetes mellitus, insulin signalling, mitogen-activated protein kinase signalling) and T cell development, regulation and activation (T cell receptor signalling), as well as transcription factors (TCF3, LEF1 and NFATC) that regulate T cells. Furthermore, bump-hunting analysis revealed differentially methylated regions in PRDM16 and HLA-DPB1, genes important for adipose tissue differentiation, stem cell maintenance and function and T cell activation. Conclusions/interpretation Our findings suggest that the alterations in methylation patterns observed in IUGR CD3 + T cells may have functional consequences in targeted genes, regulatory regions and transcription factors. These may serve as biomarkers to identify those at 'high risk' for diminished attainment of full health potential who can benefit from early interventions.

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“…Not only are these techniques considered superior in determining methylation status, they are also more cost and time efficient for the analysis of tissue biomarkers as diagnostic tools. 8 Although bisulphite sequencing represents the gold standard for finding differentially methylated DNA sequences at single-nucleotide resolution, currently both whole genome and genome-wide bisulphite sequencing techniques are considered too costly for applications with larger sample numbers. 9,10 As an alternative, the BeadArray technique, designed by Illumina, has been widely used to interrogate DNA-methylation in a genome-wide manner, although with less coverage than genome-wide bisulphite sequencing.…”

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confidence: 99%

Formalin-fixed, paraffin-embedded (FFPE) tissue epigenomics using Infinium HumanMethylation450 BeadChip assays

Ruijter

Hoon

Slaats

et al. 2015

Laboratory Investigation

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Current genome-wide methods to detect DNA-methylation in healthy and diseased tissue require high-quality DNA from fresh-frozen (FF) samples. However, well-annotated clinical samples are mostly available as formalin-fixed, paraffinembedded (FFPE) tissues containing poor-quality DNA. To overcome this limitation, we here aimed to evaluate a DNA restoration protocol for usage with the genome-wide Infinium HumanMethylation450 BeadChip assay (HM-450K). Sixty-six DNA samples from normal colon (n = 9) and breast cancer (n = 11) were interrogated separately using HM-450K. Analyses included matched FF/FFPE samples and technical duplicates. FFPE DNA was processed with (FFPEr) or without a DNA restoration protocol (Illumina). Differentially methylated genes were finally validated in 24 additional FFPE tissues using nested methylation-specific PCR (MSP). In summary, β-values correlation between FFPEr duplicates was high (ρ = 0.9927 (s.d. ± 0.0015)). Matched FF/FFPEr correlation was also high (ρ = 0.9590 (s.d. ± 0.0184)) compared with matched FF/FFPE (ρ = 0.8051 (s.d. ± 0.1028). Probe detection rate in FFPEr samples (98.37%, s.d. ± 0.66) was comparable to FF samples (99.98%, s.d. ± 0.019) and substantially lower in FFPE samples (82.31%, s.d. ± 18.65). Assay robustness was not decreased by sample archival age up to 10 years. We could also demonstrate no decrease in assay robustness when using 100 ng of DNA input only. Four out of the five selected differentially methylated genes could be validated by MSP. The gene failing validation by PCR showed high variation of CpG β-values in primer-binding sites. In conclusion, by using the FFPE DNA restoration protocol, HM-450K assays provide robust, accurate and reproducible results with FFPE tissue-derived DNA, which are comparable to those obtained with FF tissue. Most importantly, differentially methylated genes can be validated using more sensitive techniques, such as nested MSP, altogether providing an epigenomics platform for molecular pathological epidemiology research on archived samples with limited tissue amount. Epigenomic changes are recognised as important factors in tumour initiation, growth and progression. Global and local DNA-methylation patterns are frequently altered in cancer cells, resulting in genomic instability and diminished or elevated expression of tumour-suppressor genes or oncogenes, respectively, driving malignant transformation, growth promotion and metastasis (reviewed in Jones and Baylin 1 and Petronis 2 ). In the clinical setting, cancer-specific DNA-methylation changes are increasingly evaluated as biomarkers for early detection, staging or patient prognosis.In addition, a role for DNA-methylation changes in mediating either cancer drug resistance or drug sensitivity has been hypothesised and verified. [3][4][5][6] Therefore, the in-depth study of the cancer DNA-methylome holds promise to provide important clues as to which genes and biological networks are affected at tumour initiation and progression. Furthermore, it will provide clues as to which g...

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Recommendations for the design and analysis of epigenome-wide association studies

Cited by 352 publications

References 51 publications

Epigenome-wide association study reveals longitudinally stable DNA methylation differences in CD4+ T cells from children with IgE-mediated food allergy

Epigenome-wide association study reveals longitudinally stable DNA methylation differences in CD4+ T cells from children with IgE-mediated food allergy

DNA hypermethylation of CD3+ T cells from cord blood of infants exposed to intrauterine growth restriction

Formalin-fixed, paraffin-embedded (FFPE) tissue epigenomics using Infinium HumanMethylation450 BeadChip assays

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