Detection of Loss of Imprinting by Pyrosequencing®
Part of the
Methods in Molecular Biology
book series (MIMB, volume 1315)
Genomic imprinting is an epigenetically regulated process determining allele-specific expression in a parent-of-origin dependent manner. Altered expression of imprinted genes characterizes numerous congenital diseases including Beckwith–Wiedemann, Silver–Russell, Angelman, and Prader–Willi syndromes as well as acquired disorders such as cancer. The detection of imprinting alterations has important translational implications in clinics and the application of the Pyrosequencing® technology offers the possibility to identify accurately also subtle modifications in allele-specific expression and in DNA methylation levels.
Here, we describe two methods to investigate genomic imprinting defects (loss of imprinting, LOI) using Pyrosequencing: (1) Allele-specific expression analysis based on single nucleotide polymorphism (SNP), and (2) quantification of DNA methylation.
The protocol for the quantification of the allele-specific expression is carried out by analyzing an informative SNP located within the transcribed portion of an imprinted gene. The method includes the cDNA amplification of the region containing the SNP and the Pyrosequencing-based analysis for the quantitative allelic discrimination comparing the ratio of the two alleles.
The second protocol allows the accurate quantification of the DNA methylation levels at the Imprinting Control Regions (ICRs). Imprinted genes are clustered in chromosomal regions and their expression is mainly regulated by DNA methylation at CpG sites located within the ICRs. After bisulfite modification of the genomic DNA, the region of interest is amplified by PCR and analyzed by Pyrosequencing. The methylation value at each CpG site is calculated by the CpG software, which determines the ratio of the incorporation of “C” and “T” and converts the value in methylation percentage.
Key wordsGenomic imprinting Epigenetics DNA methylation Pyrosequencing® Allele-specific expression Loss of imprinting ICR Prader–Willi syndrome Angelman syndrome Beckwith–Wiedemann syndrome Silver–Russell syndrome
Feinberg AP, Cui H, Ohlsson R (2002) DNA methylation and genomic imprinting: insights from cancer into epigenetic mechanisms. Sem Cancer Biol 12:389–398CrossRefGoogle Scholar
Rainier S, Johnson LA, Dobry CJ et al (1993) Relaxation of imprinted genes in human cancer. Nature 362:747–749PubMedCrossRefGoogle Scholar
Djiogue S, Nwabo Kamdje AH, Vecchio L et al (2013) Insulin resistance and cancer: the role of insulin and IGFs. Endocr Relat Cancer 20:R1–R17PubMedCrossRefGoogle Scholar
Delaval K, Wagschal A, Feil R (2006) Epigenetic deregulation of imprinting in congenital diseases of aberrant growth. Bioessays 28:453–459PubMedCrossRefGoogle Scholar
Tabano S, Colapietro P, Cetin I et al (2010) Epigenetic modulation of the IGF2/H19 imprinted domain in human embryonic and extra-embryonic compartments and its possible role in fetal growth restriction. Epigenetics 5:313–324PubMedCrossRefGoogle Scholar
Dejeux E, El abdalaoui H, Gut IG et al (2009) Identification and quantification of differentially methylated loci by the pyrosequencing technology. Methods Mol Biol 507:189–205PubMedGoogle Scholar
Kantor B, Shemer R, Razin A (2006) The Prader-Willi/Angelman imprinted domain and its control center. Cytogenet Genome Res 113:300–305PubMedCrossRefGoogle Scholar
Glenn CC, Saitoh S, Jong MT et al (1996) Gene structure, DNA methylation, and imprinted expression of the human SNRPN gene. Am J Hum Genet 58:335–346PubMedCentralPubMedGoogle Scholar
Kubota T, Sutcliffe JS, Aradhya S et al (1996) Validation studies of SNRPN methylation as a diagnostic test for Prader-Willi syndrome. Am J Med Genet 66:77–80PubMedCrossRefGoogle Scholar
Glenn CC, Driscoll DJ, Yang TP et al (1997) Genomic imprinting: potential function and mechanisms revealed by the Prader-Willi and Angelman syndromes. Mol Hum Reprod 3:321–332PubMedCrossRefGoogle Scholar
Horsthemke B, Wagstaff J (2008) Mechanisms of imprinting of the Prader-Willi/Angelman region. Am J Med Genet A 146A:2041–2052PubMedCrossRefGoogle Scholar
Calvello M, Tabano S, Colapietro P et al (2013) Quantitative DNA methylation analysis improves epigenotype-phenotype correlations in Beckwith-Wiedemann syndrome. Epigenetics 8:1053–1060PubMedCentralPubMedCrossRefGoogle Scholar
Eggermann T, Eggermann K, Schönherr N (2008) Growth retardation versus overgrowth: Silver-Russell syndrome is genetically opposite to Beckwith-Wiedemann syndrome. Trends Genet 24:195–204PubMedCrossRefGoogle Scholar
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