Schematic structure of studied imprinted genes on mouse chromosome 7q.

<p>Proximally, near the centromere, are <i>Paternally expressed gene 3 (Peg3)</i> and <i>Small Nuclear Ribonucleoprotein Polypeptide N (Snrpn)</i>, and distally <i>Insulin-like growth factor 2</i>/<i>H19 locus (Igf2/H19)</i>. Gray boxes illustrate maternally or paternally expressed active alleles and black boxes inactive alleles. Imprinting control regions (ICR) or differentially methylated regions (DMR) are marked with methylated (black) or unmethylated (white) balls. Arrows and sequences below the ICRs or DMR represent the regions of interest. Not drawn into scale.</p

Expression levels of imprinted genes in control and alcohol-exposed 9.5 embryonic day old (E9.5) embryos and placentas as well as E16.5 placentas.

<p><i>Igf2</i>, <i>H19</i>, <i>Snrpn</i> and <i>Peg3</i> expressions in control and alcohol-exposed E9.5 embryos (A), E9.5 placentas (B) and in E16.5 placentas (C). Control samples are colored in black and alcohol-exposed samples in gray. The numbers of samples are presented in columns (expression of <i>Snrpn</i> in one embryo and one E9.5 placenta samples was not detected). Error bars denote the SD. P-value; (A) two-way Student’s t-test: *p<0.05, **p<0.001, (C) Two-way ANOVA: *p<0.05.</p

Altered volumes of left and right hippocampi, olfactory bulbs, and lateral ventricles of ethanol-exposed offspring.

<p>Wilcoxon test was used to access left-right differences within the same animals (#) and Mann-Whitney test to compare control and ethanol-exposed (EtOH) mice (*). ^#/*p<0.05 and ^###p<0.001. Each dot represents an individual adult male mouse (P60). Control mice are illustrated in black and ethanol-exposed offspring in grey. Bars are averaged values ± SD.</p

Site-specific DNA methylation levels of six CpG islands in control and ethanol-exposed offspring (P28).

<p>Site-specific methylation levels of <i>Vmn2r64</i> [A: upstream island (1) and B: CpG island in gene-body (2)], <i>Olfr110</i> (C, upstream), <i>Vpreb2</i> (D, in gene-body), <i>and Olfr601</i> (E, in gene-body) in hippocampus (HC) and <i>Olfr601</i> (F, in gene-body) in main olfactory epithelium (MOE). The methylation levels of CpG1 in <i>Vmn2r64</i> (1)(A), CpG3 in <i>Olfr110</i> (C), and CpG1 in <i>Olfr601</i> (E) were significantly changed between the two experimental groups (*p<0.05, Mann-Whitney). Notable changes are marked by diamonds (♦): CpG4 and CpG5 in <i>Olfr110</i> (p = 0.07 and p = 0.11, Mann-Whitney, respectively). Controls are illustrated in white and ethanol-exposed offspring in grey bars.</p

DNA methylation levels of CpG islands of five candidate genes in control and ethanol-exposed offspring.

<p>DNA methylation levels of CpG islands in <i>Vmn2r64</i> [A: upstream CpG island (1) and B: CpG island in gene-body (2)], <i>Olfr110</i> (C, upstream), <i>Vpreb2</i> (D, in gene-body), <i>and Olfr601</i> (E, in gene-body) in hippocampus (HC) and <i>Olfr601</i> (F, in gene-body) in main olfactory epithelium (MOE). Each dot represents an average of methylation percent of clones in a particular CpG island from individual control or ethanol-exposed (EtOH) male offspring (P28).</p

Altered gene expression of 23 genes and three microRNAs in hippocampus of ethanol-exposed offspring (P28).

<p>Nominal p-values <0.05, FC ≥ 1.5.</p><p>Altered gene expression of 23 genes and three microRNAs in hippocampus of ethanol-exposed offspring (P28).</p

Effects of gestational alcohol exposure on gene expression in main olfactory epithelium and bone marrow.

<p>Quantitative PCR studies showed increased expression of <i>Olfr601</i> in main olfactory epithelium (MOE) and <i>Vpreb2</i> in bone marrow (*p<0.05 and ***p<0.001, respectively, one-tailed Student’s t-test) of ethanol-exposed (EtOH) offspring relative to reference gene <i>Rps16</i>. Expression of <i>H2-M10</i>.<i>3</i> was not significantly changed in MOE (p = 0.35, one-tailed Student’s t-test). Each dot represents an individual four-week-old (P28) male mouse. Bars are averaged values ± SD.</p

Additional file 1: of rs10732516 polymorphism at the IGF2/H19 locus associates with genotype-specific effects on placental DNA methylation and birth weight of newborns conceived by assisted reproductive technology

Table S1. Information of ART samples. (PDF 165 kb

Additional file 2: of rs10732516 polymorphism at the IGF2/H19 locus associates with genotype-specific effects on placental DNA methylation and birth weight of newborns conceived by assisted reproductive technology

Table S1. DNA methylation levels of H19 ICR (CTCF6), H19 DMR and LINE-1 in control and ART-derived placentas by EpiTYPER method. Methylation average values with SDs (±) of CpG units are presented. Gray boxes present significant methylation level difference at CpG sites in H19 DMR between ART and control samples within A/A genotype (nominal p-value < 0.05, Student’s t-test). Table S2. Total, rs10732516 genotype-specific (patG/matA and patA/matG) and allele-specific (paternal and maternal) DNA methylation levels at H19 ICR (CTCF6) in control and ART-derived placentas by traditional bisulfite sequencing. Gray boxes present significant methylation level difference at CpG sites between control and ART samples within genotypes and alleles (nominal p-value < 0.05, Mann-Whitney). Total methylation levels: total means of both genotypes and both alleles are included. Genotype-specific methylation levels: total means of both alleles are included. Allele-specific methylation levels: maternal and paternal alleles are presented separately. Table S3. rs10732516 patA/matG genotype- and allele-specific DNA methylation levels at H19 ICR1 (CTCF6) in controls and ART-derived newborns’ white blood cells (WBCs) in cord blood by traditional bisulfite sequencing. Genotype-specific methylation levels: total means of both alleles are included. Allele-specific methylation levels: maternal and paternal alleles are presented separately. (PDF 145 kb

Additional file 3: of rs10732516 polymorphism at the IGF2/H19 locus associates with genotype-specific effects on placental DNA methylation and birth weight of newborns conceived by assisted reproductive technology

Protocol S1. Extraction of total white blood cells (WBC) from EDTA-tube. Table S2. Primers (A) and PCR reactions (B). (PDF 306 kb