title: Quantification of the methylation status of the PWS/AS imprinted region: comparison of two approaches based on bisulfite sequencing and methylation-sensitive MLPA.
abstract: Standard methods used for genomic methylation analysis allow the detection of complete absence of either methylated or non-methylated alleles but are usually unable to detect changes in the proportion of methylated and unmethylated alleles. We compare two methods for quantitative methylation analysis, using the chromosome 15q11-q13 imprinted region as model. Absence of the non-methylated paternal allele in this region leads to Prader-Willi syndrome (PWS) whilst absence of the methylated maternal allele results in Angelman syndrome (AS). A proportion of AS is caused by mosaic imprinting defects which may be missed with standard methods and require quantitative analysis for their detection. Sequence-based quantitative methylation analysis (SeQMA) involves quantitative comparison of peaks generated through sequencing reactions after bisulfite treatment. It is simple, cost-effective and can be easily established for a large number of genes. However, our results support previous suggestions that methods based on bisulfite treatment may be problematic for exact quantification of methylation status. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) avoids bisulfite treatment. It detects changes in both CpG methylation as well as copy number of up to 40 chromosomal sequences in one simple reaction. Once established in a laboratory setting, the method is more accurate, reliable and less time consuming.
authors.affiliation: Institute of Human Genetics, University of Heidelberg, Im Neuenheimer Feld 366, D-69120 Heidelberg, Amsterdam, The Netherlands.
authors.forename: Nicola
authors.initials: N
authors.lastname: Dikow
authors.forename: Anders Oh
authors.initials: AO
authors.lastname: Nygren
authors.forename: Jan P
authors.initials: JP
authors.lastname: Schouten
authors.forename: Carolin
authors.initials: C
authors.lastname: Hartmann
authors.forename: Nikola
authors.initials: N
authors.lastname: Krämer
authors.forename: Bart
authors.initials: B
authors.lastname: Janssen
authors.forename: Johannes
authors.initials: J
authors.lastname: Zschocke
mesh_terms: D017204:Angelman Syndrome / Q000235:genetics*; D016022:Case-Control Studies; D019175:DNA Methylation / Q000187:drug effects; D018392:Genomic Imprinting; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011218:Prader-Willi Syndrome / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites / Q000494:pharmacology*
chemical_list: D013447:Sulfites; C042345:hydrogen sulfite