Hypermethylation of the non-imprinted maternal MEG3 and paternal MEST alleles is highly variable among normal individuals

Imprinted genes show parent-specific activity (functional haploidy), which makes them particularly vulnerable to epigenetic dysregulation. Here we studied the methylation profiles of oppositely imprinted genes at single DNA molecule resolution by two independent parental allele-specific deep bisulfite sequencing (DBS) techniques. Using Roche (GSJunior) next generation sequencing technology, we analyzed the maternally imprinted MEST promoter and the paternally imprinted MEG3 intergenic (IG) differentially methylated region (DMR) in fetal cord blood, adult blood, and visceral adipose tissue. Epimutations were defined as paternal or maternal alleles with >50% aberrantly (de)methylated CpG sites, showing the wrong methylation imprint. The epimutation rates (range 2-66%) of the paternal MEST and the maternal MEG3 IG DMR allele, which should be completely unmethylated, were significantly higher than those (0-15%) of the maternal MEST and paternal MEG3 alleles, which are expected to be fully methylated. This hypermethylation of the non-imprinted allele (HNA) was independent of parental origin. Very low epimutation rates in sperm suggest that HNA occurred after fertilization. DBS with Illumina (MiSeq) technology confirmed HNA for the MEST promoter and the MEG3 IG DMR, and to a lesser extent, for the paternally imprinted secondary MEG3 promoter and the maternally imprinted PEG3 promoter. HNA leads to biallelic methylation of imprinted genes in a considerable proportion of normal body cells (somatic mosaicism) and is highly variable between individuals. We propose that during development and differentiation maintenance of differential methylation at most imprinting control regions may become to some extent redundant. The accumulation of stochastic and environmentally-induced methylation errors on the non-imprinted allele may increase epigenetic diversity between cells and individuals.