Additional file 1: Table S1. of Epigenetic signatures of gestational diabetes mellitus on cord blood methylation

Primers for pyrosequencing. (DOC 52 kb

Additional file 4: Table S3. of Epigenetic signatures of gestational diabetes mellitus on cord blood methylation

Multivariate analyses (adjusting for maternal BMI, gestational age, and fetal sex): CpG methylation of candidate genes in GDM versus control FCB samples. (DOC 68 kb

Additional file 2: Figure S1. of Epigenetic signatures of gestational diabetes mellitus on cord blood methylation

Estimation of blood cell composition based on 450K methylation array profiles. Blue box plots show the distribution of cell types in GDM cord blood and red box plots in control samples. The median is represented by a horizontal line. The bottom of the box indicates the 25th percentile and the top the 75th percentile. Outliers are shown as circles. (DOC 146 kb

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

<div><p>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 <i>MEST</i> promoter and the paternally imprinted <i>MEG3</i> 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 <i>MEST</i> and the maternal <i>MEG3</i> IG DMR allele, which should be completely unmethylated, were significantly higher than those (0–15%) of the maternal <i>MEST</i> and paternal <i>MEG3</i> 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 <i>MEST</i> promoter and the <i>MEG3</i> IG DMR, and to a lesser extent, for the paternally imprinted secondary <i>MEG3</i> promoter and the maternally imprinted <i>PEG3</i> 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.</p></div

Mean FCB methylation and epimutation rates of maternal versus paternal alleles of the <i>MEG3</i> IG DMR, <i>MEG3</i> promoter, <i>MEST</i>, and <i>PEG3</i>.

<p>DBS was performed with the Illumina MiSeq. The non-imprinted allele of <i>MEG3</i> (both primary and secondary DMR) and <i>MEST</i> exhibited significantly higher epimutation rates than the imprinted allele.</p

Parental allele-specific methylation of the <i>MEG3</i> IG DMR and the <i>MEST</i> promoter.

<p>Mean methylation levels and standard deviations of the paternal vs. maternal alleles were determined by DBS with the Roche GSJunior in FCB, AB, and VAT. For both genes, the non-imprinted allele, which is expected to be completely unmethylated, showed an aberrantly high methylation in all analyzed tissues, whereas the imprinted allele showed the expected (90–100%) methylation.</p

Parental allele-specific epimutation rates of the <i>MEG3</i> IG DMR and the <i>MEST</i> promoter.

<p>The percentage of alleles with >50% aberrantly (de)methylated CpGs was demined by DBS with the Roche GSJunior in FCB, AB, and VAT samples. The unmethylated alleles of the paternally imprinted <i>MEG3</i> and the maternally imprinted <i>MEST</i> genes displayed significantly higher epimutation rates than the methylated alleles.</p

Epimutation rates of the <i>MEG3</i> IG DMR and the <i>MEST</i> promoter in individual samples.

<p>The percentage of paternal (black dots) versus maternal alleles (gray dots) with >50% aberrantly (de)methylated CpGs was determined by DBS with the Roche GSJunior in individual FCB, AB, and VAT samples. With very few exceptions, the unmethylated alleles of the paternally imprinted <i>MEG3</i> and the maternally imprinted <i>MEST</i> genes displayed much higher epimutation rates than the methylated alleles.</p