CpG Islands pp 109-121 | Cite as

The Use of Methylation-Sensitive Multiplex Ligation-Dependent Probe Amplification for Quantification of Imprinted Methylation

  • Ana Monteagudo-Sánchez
  • Intza Garin
  • Guiomar Perez de Nanclares
  • David MonkEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1766)


Imprinting disorders are a group of congenital diseases that can result from multiple mechanisms affecting imprinted gene dosage including cytogenetic aberration and epigenetic anomalies. Quantification of CpG methylation and correct copy-number calling is required for molecular diagnosis. Methylation-sensitive multiplex ligation-dependent probe amplification (MS-MLPA) is a multiplex method that accurately measures both parameters in a single assay. This technique relies upon the ligation of MLPA probe oligonucleotides and digestion of the genomic DNA–probe hybrid complexes with the Hha1 methylation-sensitive restriction endonuclease prior to fluorescent PCR amplification with a single primer pair. Since each targeted probe contains stuffer sequence of varying length, each interrogated position is visualized as an amplicon of different size upon capillary electrophoresis.

Key words

Imprinting DNA methylation Methylation-sensitive multiplex ligation-dependent probe amplification 



This work was supported by Spanish Ministry of Economy and Competitiveness (MINECO) (BFU2014-53093-R to D.M) cofunded with the European Union Regional Development Fund (FEDER); Institute of Health Carlos III of the Ministry of Economy and Competitiveness cofinanced with European Union ERDF funds (PI13/00467 to G.P.dN.); the Basque Department of Health (GV2014111017 to G.P.dN.) and the I3SNS Program of the Spanish Ministry of Health (CP03/0064; S.I.V.I. 1395/09 to G.P.dN.). AMS is a recipient of an F.P.I. Ph.D. studentship from MINECO. All authors are members of the “European Network for Human Congenital Imprinting Disorders” COST action (BM1208).


  1. 1.
    Soellner L, Begemann M, Mackay DJ et al (2016) Recent advances in imprinting disorders. Clin Genet. CrossRefGoogle Scholar
  2. 2.
    Monk D (2015) Germline-derived DNA methylation and early embryo epigenetic reprogramming: the selected survival of imprints. Int J Biochem Cell Biol 67:128–138. CrossRefPubMedGoogle Scholar
  3. 3.
    Nygren AO, Ameziane N, Duarte HM et al (2005) Methylation-specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences. Nucleic Acids Res 33:e128. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Procter M, Chou LS, Tang W et al (2006) Molecular diagnosis of Prader-Willi and Angelman syndromes by methylation-specific melting analysis and methylation-specific multiplex ligation-dependent probe amplification. Clin Chem 52:1276–1283. CrossRefPubMedGoogle Scholar
  5. 5.
    Dawson AJ, Cox J, Hovanes K et al (2015) PWS/AS MS-MLPA confirms maternal origin of 15q11.2 microduplication. Case Rep Genet 2015:474097. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Priolo M, Sparago A, Mammì C et al (2008) MS-MLPA is a specific and sensitive technique for detecting all chromosome 11p15.5 imprinting defects of BWS and SRS in a single-tube experiment. Eur J Hum Genet 16:565–571. CrossRefPubMedGoogle Scholar
  7. 7.
    Scott RH, Douglas J, Baskcomb L et al (2008) Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) robustly detects and distinguishes 11p15 abnormalities associated with overgrowth and growth retardation. J Med Genet 45:106–113. CrossRefPubMedGoogle Scholar
  8. 8.
    Garin I, Mantovani G, Aguirre U et al (2015) European guidance for the molecular diagnosis of pseudohypoparathyroidism not caused by point genetic variants at GNAS: an EQA study. Eur J Hum Genet 23:560. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Yuno A, Usui T, Yambe Y et al (2013) Genetic and epigenetic states of the GNAS complex in pseudohypoparathyroidism type Ib using methylation-specific multiplex ligation-dependent probe amplification assay. Eur J Endocrinol 168:169–175. CrossRefPubMedGoogle Scholar
  10. 10.
    Sachwitz J, Strobl-Wildemann G, Fekete G et al (2016) Examinations of maternal uniparental disomy and epimutations for chromosomes 6, 14, 16 and 20 in Silver-Russell syndrome-like phenotypes. BMC Med Genet 17:20. PMID: 26969265CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Price EA, Price K, Kolkiewicz K et al (2014) Spectrum of RB1 mutations identified in 403 retinoblastoma patients. J Med Genet 51:208–214. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Ana Monteagudo-Sánchez
    • 1
  • Intza Garin
    • 2
  • Guiomar Perez de Nanclares
    • 2
  • David Monk
    • 1
    Email author
  1. 1.Imprinting and Cancer Group, Cancer Epigenetic and Biology Program (PEBC), Bellvitge Institute for Biomedical Research (IDIBELL)Hospital Duran and ReynalsBarcelonaSpain
  2. 2.Molecular (Epi)Genetics Laboratory, BioAraba National Health InstituteOSI Araba-TxagorritxuVitoria-GasteizSpain

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