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

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

Analysis of <i>ROBO1</i> in the Large Family Linked to <i>DYX5</i>

<div><p>(A) An abridged pedigree of the family linked to <i>DYX5</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010050#pgen-0010050-b17" target="_blank">17</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010050#pgen-0010050-b19" target="_blank">19</a>]. Numbers refer to samples studied for <i>ROBO1</i> expression (C and D). A dot indicates carriers of the dyslexia-linked haplotype [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010050#pgen-0010050-b17" target="_blank">17</a>] and circled dots indicate individuals genotyped for all markers (B). Diamonds denote individuals genotyped for all markers, but not sharing the haplotype. Affected individuals are shaded black and unverified dyslectics are shaded gray.</p><p>(B) Markers (right) and alleles (left) that define the haplotype linked to dyslexia (A). The bar indicates the extent of the <i>ROBO1</i> haplotype carried by patients marked with a dot (A).</p><p>(C) Sequencing of cDNA reveals absent or attenuated expression (<i>p</i> < 0.017 for all measurements) of the <i>ROBO1</i> allele (SNP 6483A > T indicated by arrows) encoded by the dyslexia-linked haplotype as compared to genomic sequence. In the control, both alleles show equal allelic ratios in genomic and cDNA. Patient numbers refer to (A).</p><p>(D) Attenuation of <i>ROBO1</i> mRNA expression from the dyslexia-associated allele. Allelic expression of <i>ROBO1</i> was assessed by sequencing the SNP 6483 (A/T) as in (C). Allelic ratios were assessed by five to six replicated sequencing tracings in four controls (21 data points) and four dyslexic individuals (24 data points). The results are expressed as the mRNA level of the dyslexia-associated allele as compared to the corresponding allele mRNA level in controls. Data are shown as mean ± 1 standard error of the mean (bars). Significance was assessed by two-tailed <i>t</i> test.</p></div

Coding Changes of <i>ROBO1</i> during Primate Evolution

<p>Phylogenetic tree of ROBO1 protein evolution in hominoids. Rat was used as the out-group in sequence comparisons. dN/dS ratios of the branches were calculated with the Codeml program, assuming a freely varying ratio. A model in which omega value was higher in lineages leading to humans, chimpanzees, and gorillas was significantly better than a free-ratio model (<i>p</i> < 0.001).</p

Delineation of Translocation Breakpoint Region and <i>ROBO1</i> Structure

<div><p>(A) Fluorescence in situ hybridization with BAC clone RP11-143B12 as a probe, showing hybridization signals in Chromosome 3 (upward arrow), der(3), and der(8) (horizontal arrows).</p><p>(B) Southern hybridization with a probe derived from RP11-143B12 shows genomic rearrangements (arrowheads) with five restriction enzymes in translocation patient (P) compared to the control sample.</p><p>(C) A gene map of Chromosome 3p13-3q11.1 showing the cytogenetic localization of the translocation breakpoint (black bar). An arrow indicates the direction of <i>ROBO1</i> transcription. Localization of the translocation breakpoint (square bracket) to BACs AC117479 and AC117461.</p><p>(D) Splice variants and exon structure of <i>ROBO1</i> (exons numbered from 1–30). Novel exons a and 7b and additional sequence to exon 1 are indicated in solid black. Exons unique to <i>ROBO1</i> (hatched black) and <i>DUTT1</i> (hatched grey) and common to both <i>ROBO1</i> and <i>DUTT1</i> (solid grey) are indicated. Corresponding BACs to exons are shown below. The translocation disrupting <i>ROBO1</i> between exons 1 and 2 in AC117479 is shown by vertical grey bar. Dotted lines indicate <i>DUTT1</i> variants. Novel splice variants are shown by grey lines and numbered (1), exclusion of exon 2 (89–169 of AF040990) (2), exclusion of <i>DUTT1</i> exon 2 (1019–1345 of Z95705) (3), exclusion of exon 19 (2813–2829 of AF040990) (4), initial 165 bp of exon 22 (3037–3201 of AF040990) (5), 905 bp from exons 24–28 (3603–4508 of AF040990) (6), 878 bp from exons 25–28 (3641–4528 of AF040990) and (7), exclusion of exon 29 (4745–4939 of AF040990).</p></div

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