Multilocus Methylation Assays in Epigenetics

  • Thomas Eggermann
Reference work entry


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.


5-Hydroxymethyl cytosine DNA methylation Enrichment-restriction enzyme Affinity enrichment Bisulfite conversion Array hybridization Next-generation sequencing MLPA Pyrosequencing 

List of Abbreviations










Base pair


Chromatin immunoprecipitation


Copy number variation


Combined bisulfite restriction analysis




Differentially methylated region


DNA methyltransferase


Multiplex ligation probe-dependent amplification


Methylation specific


Methylation-sensitive restriction enzyme PCR


Noncoding RNA


Next-generation sequencing


Polymerase chain reaction


Quantitative analysis of methylated alleles


Restriction endonuclease


Reduced representation bisulfite sequencing


Sequencing by ligation


Sequencing by synthesis


Ten-eleven translocation methylcytosine dioxygenase


Uniparental disomy


  1. Alders M, Maas SM, Kadouch DJ et al (2014) Methylation analysis in tongue tissue of BWS patients identifies the (EPI) genetic cause in 3 patients with normal methylation levels in blood. Eur J Med Genet 57:293–297CrossRefGoogle Scholar
  2. Barski A, Cuddapah S, Cui K et al (2007) High-resolution profiling of histone methylations in the human genome. Cell 129:823–837CrossRefGoogle Scholar
  3. Beygo J, Ammerpohl O, Gritzan D et al (2013) Deep bisulfite sequencing of aberrantly methylated loci in a patient with multiple methylation defects. PLoS One 9:e76953CrossRefGoogle Scholar
  4. Bibikova M, Barnes B, Tsan C et al (2011) High density DNA methylation array with single CpG site resolution. Genomics 98:288–295CrossRefGoogle Scholar
  5. Bochtler M, Kolano A, Xu GL (2017) DNA demethylation pathways: additional players and regulators. BioEssays 39:1–13CrossRefGoogle Scholar
  6. Docherty LE, Rezwan FI, Poole RL et al (2015) Mutations in NLRP5 are associated with reproductive wastage and multilocus imprinting disorders in humans. Nat Commun 6:8086. Scholar
  7. Eads CA, Danenberg KD, Kawakami K et al (2000) MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res 28:E32CrossRefGoogle Scholar
  8. Eggermann K, Bliek J, Brioude F et al (2016) EMQN best practice guidelines for the molecular genetic testing and reporting of chromosome 11p15 imprinting disorders: Silver-Russell and Beckwith-Wiedemann syndrome. Eur J Hum Genet 24:1377–1387CrossRefGoogle Scholar
  9. Enklaar T, Zabel BU, Prawitt D (2006) Beckwith-Wiedemann syndrome: multiple molecular mechanisms. Expert Rev Mol Med 8:1–19CrossRefGoogle Scholar
  10. Frommer M, McDonald LE, Millar DS et al (1992) A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci U S A 89:1827–1831CrossRefGoogle Scholar
  11. Goodwin S, McPherson JD, McCombie WR (2016) Coming of age: ten years of next-generation sequencing technologies. Nat Rev Genet 17:333–351CrossRefGoogle Scholar
  12. Gu H, Bock C, Mikkelsen TS, Jäger N et al (2010) Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution. Nat Methods 7:133–136CrossRefGoogle Scholar
  13. Heijmans BT, Tobi EW, Stein AD et al (2008) Persistent epigenetic differences associated with prenatal exposure to famine in humans. Proc Natl Acad Sci U S A 105:17046–17049CrossRefGoogle Scholar
  14. Holliday R (1990) DNA methylation and epigenetic inheritance. Philos Trans R Soc Lond Ser B Biol Sci 326:329–338CrossRefGoogle Scholar
  15. Ibanez de Caceres I, Dulaimi E, Hoffman AM et al (2006) Identification of novel target genes by an epigenetic reactivation screen of renal cancer. Cancer Res 66:5021–5028CrossRefGoogle Scholar
  16. Kelsey G, Feil R (2013) New insights into establishment and maintenance of DNA methylation imprints in mammals. Philos Trans R Soc Lond Ser B Biol Sci 368:20110336. Scholar
  17. Killian JK, Bilke S, Davis S et al (2009) Large-scale profiling of archival lymph nodes reveals pervasive remodeling of the follicular lymphoma methylome. Cancer Res 69:758–764CrossRefGoogle Scholar
  18. Laird PW (2010) Principles and challenges of genomewide DNA methylation analysis. Nat Rev Genet 1(1):191–203CrossRefGoogle Scholar
  19. Mackay DJ, Callaway JL, Marks SM et al (2008) Hypomethylation of multiple imprinted loci in individuals with transient neonatal diabetes is associated with mutations in ZFP57. Nat Genet 40:949–951CrossRefGoogle Scholar
  20. McCarrey JR, Lehle JD, Raju SS (2016) Tertiary epimutations – a novel aspect of epigenetic transgenerational inheritance promoting genome instability. PLoS One 11(12):e0168038Google Scholar
  21. McCullough LE, Miller EE, Mendez MA et al (2016) Maternal B vitamins: effects on offspring weight and DNA methylation at genomically imprinted domains. Clin Epigenetics 8:8. Scholar
  22. Melnikov AA, Gartenhaus RB, Levenson AS et al (2005) MSRE-PCR for analysis of gene-specific DNA methylation. Nucleic Acids Res 33:e93CrossRefGoogle Scholar
  23. Monk D, Morales J, den Dunnen JT, et al (2016) Recommendations for a nomenclature system for reporting methylation aberrations in imprinted domains. Epigenetics [Epub ahead of print]Google Scholar
  24. Paul CL, Clark SJ (1996) Cytosine methylation: quantitation by automated genomic sequencing and GENESCAN analysis. BioTechniques 21:126–133CrossRefGoogle Scholar
  25. Ronaghi M, Uhlén M, Nyrén PA (1998) Sequencing method based on real-time pyrophosphate. Science 281:363CrossRefGoogle Scholar
  26. Russo S, Calzari L, Mussa A et al (2016) A multi-method approach to the molecular diagnosis of overt and borderline 11p15.5 defects underlying Silver-Russell and Beckwith-Wiedemann syndromes. Clin Epigenetics 8:23CrossRefGoogle Scholar
  27. Schouten JP, McElgunn CJ, Waaijer R et al (2002) Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res e57:30Google Scholar
  28. Schumacher A, Kapranov P, Kaminsky Z et al (2006) Microarray-based DNA methylation profiling: technology and applications. Nucleic Acids Res 34:528–542CrossRefGoogle Scholar
  29. Shin C, Han C, Pae CU et al (2016) Precision medicine for psychopharmacology: a general introduction. Expert Rev Neurother 16(7):831–839CrossRefGoogle Scholar
  30. Soellner L, Monk D, Rezwan FI et al (2015) Congenital imprinting disorders: application of multilocus and high throughput methods to decipher new pathomechanisms and improve their management. Mol Cell Probes 29:282–290CrossRefGoogle Scholar
  31. Soellner L, Begemann M, Mackay DJ et al (2016) Recent advances in imprinting disorders. Clin Genet.
  32. Soto J, Rodriguez-Antolin C, Vallespín E et al (2016) The impact of next-generation sequencing on the DNA methylation-based translational cancer research. Transl Res 169:1–18.e1CrossRefGoogle Scholar
  33. Stuppia L, Franzago M, Ballerini P et al (2015) Epigenetics and male reproduction: the consequences of paternal lifestyle on fertility, embryo development, and children lifetime health. Clin Epigenetics 7:120CrossRefGoogle Scholar
  34. Taylor KH, Kramer RS, Davis JW et al (2007) Ultradeep bisulfite sequencing analysis of DNA methylation patterns in multiple gene promoters by 454 sequencing. Cancer Res 67:8511–8518CrossRefGoogle Scholar
  35. Uyar A, Seli E (2014) The impact of assisted reproductive technologies on genomic imprinting and imprinting disorders. Curr Opin Obstet Gynecol 26:210–221CrossRefGoogle Scholar
  36. Veeck J, Wild PJ, Fuchs T et al (2009) Prognostic relevance of Wnt-inhibitory factor-1 (WIF1) and Dickkopf-3 (DKK3) promoter methylation in human breast cancer. BMC Cancer 9:217CrossRefGoogle Scholar
  37. Xiong Z, Laird PW (1997) COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res 25:2532–2534CrossRefGoogle Scholar
  38. Zeschnigk M, Albrecht B, Buiting K et al (2008) IGF2/H19 hypomethylation in Silver-Russell syndrome and isolated hemihypoplasia. Eur J Hum Genet 16:328–334CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Institute of Human GeneticsUniversity Hospital, RWTH Technical University AachenAachenGermany

Personalised recommendations