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Handbook of Nutrition, Diet, and Epigenetics pp 2181–2202Cite as

Multilocus Methylation Assays in Epigenetics

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Abstract

Due to the basic significance of DNA methylation patterns and their changes for nearly all physiological processes, the interest in procedures to determine DNA methylation has grown rapidly and expanded. Though different levels of epigenetic regulation are known, it is advantageous to focus on DNA methylation as DNA methylation is very stable, and it is currently assumed that environmental (and inborn) factors altering epigenetic patterns mainly affect DNA methylation. Furthermore, DNA methylation tests are meanwhile well-established tools in both research and diagnostic laboratories. In the past, determination of the 5-methylcytosin and DNA methylation status was hampered because the methylation profile is not maintained during standard amplification processes. However, with the development of methylation-specific (MS) pretreatment protocols, this problem could be circumvented, and these protocols serve as pretreatment steps before the application of methylation-specific approaches. This chapter focuses on molecular tests determining the DNA methylation from single CpG to genome-wide methylation resolution. As examples for single-locus and multiloci tests, multiplex ligation probe-dependent amplification (MLPA) and pyrosequencing are described in detail, but this chapter will also introduce the potential of next-generation sequencing (NGS)-based approaches for methylation analysis. In fact NGS-based assays allow the deep and comprehensive analyses of all three mechanisms of epigenetic regulation, i.e., for histone function, analysis of (noncoding) RNA, and characterization of (differentially) methylated DNA:

  • The rapid progress in the field of DNA methylation analysis offers unique opportunities to comprehensively analyze the influence of epigenetic regulation on a broad spectrum of biological processes.

  • A broad range of methods to determine DNA methylation statuses has been developed, but the decision on the test for DNA methylation will depend on the question to be answered and the sample size which should be investigated.

  • In particular for NGS-based approaches, the running costs are currently high per sample, and bioinformatic pipelines for interpretation have to be improved, but these problems will be circumvented in the near future, and standardized software solutions will become available in the near future.

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Abbreviations

5-caC:

5-Carboxylcytosine

5-fC:

5-Formylcytosine

5-hmC:

5-Hydroxymethylcytosine

5-mC:

5-Methylcytosine

Bp:

Base pair

ChIP:

Chromatin immunoprecipitation

CNV:

Copy number variation

COBRA:

Combined bisulfite restriction analysis

CpG:

Cytosine-phosphate-guanosine

DMR:

Differentially methylated region

DNMT:

DNA methyltransferase

MLPA:

Multiplex ligation probe-dependent amplification

MS:

Methylation specific

MSRE-PCR:

Methylation-sensitive restriction enzyme PCR

ncRNA:

Noncoding RNA

NGS:

Next-generation sequencing

PCR:

Polymerase chain reaction

QAMA:

Quantitative analysis of methylated alleles

RE:

Restriction endonuclease

RRBS:

Reduced representation bisulfite sequencing

SBL:

Sequencing by ligation

SBS:

Sequencing by synthesis

TET:

Ten-eleven translocation methylcytosine dioxygenase

UPD:

Uniparental disomy

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Authors and Affiliations

  1. Institute of Human Genetics, University Hospital, RWTH Technical University Aachen, Aachen, Germany

    Thomas Eggermann

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  1. Thomas Eggermann
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Correspondence to Thomas Eggermann .

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Editors and Affiliations

  1. School of Life Sciences, University of Westminster, London, UK

    Vinood B. Patel

  2. Diabetes and Nutritional Sciences Research Division, Faculty of Life Sciences and Medicine, King’s College London, London, UK

    Victor R. Preedy

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Eggermann, T. (2019). Multilocus Methylation Assays in Epigenetics. In: Patel, V., Preedy, V. (eds) Handbook of Nutrition, Diet, and Epigenetics. Springer, Cham. https://doi.org/10.1007/978-3-319-55530-0_50

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  • DOI: https://doi.org/10.1007/978-3-319-55530-0_50

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  • Publisher Name: Springer, Cham

  • Print ISBN: 978-3-319-55529-4

  • Online ISBN: 978-3-319-55530-0

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