Additional file 1: of Genetic analysis of Wnt/PCP genes in neural tube defects

Table S1. Demographic characteristics in NTD cohorts. (DOCX 17 kb

Hypermethylated <i>H19</i> DMR1 and stably methylated <i>IGF2</i> DMR0 in human fetuses with NTDs.

<p>The DNA methylation levels of the <i>H19</i> DMR1 (A) and <i>IGF2</i> DMR0 (B) were examined through matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. In the upper panel in (A) and right panel in (B), the methylation status of all detected CpG sites was visualized. Each solid circle represents a “CG” site. (A)The number in upper panel refers to the CG site(s) in the lower histogram. (B) The number in the left panel refers to the site in the right histogram. ***: <i>P</i><0.0001 (Student's <i>t</i>-test).</p

Additional file 2: of Genetic analysis of Wnt/PCP genes in neural tube defects

Figure S1. Most frequent Pfam domains affected by Wnt/PCP genes in human NTD samples. (TIFF 2976 kb

Schematic for the imprinting mechanism of <i>IGF2</i> and <i>KCNQ1</i>.

<p>In (A), the rectangle represents each gene, and the black circle is methylated DNA. In (B), the green arrow in the lower panel represents lncRNAs <i>KCNQ1OT1</i>, and the hexagon represents <i>KvDMR1</i> that is methylated or not.</p

More open chromatin structure in mice fetuses with RA-induced spina bifida.

<p>(A) The methylation level of <i>H19</i> DMR1 is stable in RA-treated mice. For details, see the legend in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113308#pone-0113308-g003" target="_blank">Fig. 3</a>. (B) and (F): The genomic profile of genes displayed in UCSC (mm9), the genomic locus of target regions in ChIP assay (black block in the gene schematic) (corresponding results shown in C and D or G and H), and the CpG island (green block underneath the gene schematic) (corresponding results shown in E and I) detected by MASSARRAY assay are shown. The number beside the block indicates the genomic location that was targeted. In the ChIP assay results (C and D or G and H), the enrichment in the y-axis represents the relative fold enrichment in which the higher enrichment in case or control is designated as 1. *: <i>P</i><0.05; **: <i>P</i><0.01; ***: <i>P</i><0.0001 (Student's <i>t</i>-test). TSS: transcription start site.</p

Wide ectopic mRNA levels of imprinted genes in mouse fetuses with RA-induced spina bifida.

<p>(<b>A and B</b>) The real-time qPCR assay results indicate the fold change in imprinted genes on each chromosome in the spinal cord tissue of E18 RA-induced mice with NTDs compared with control. The x-axis represents the chromosome and the relative genomic position of each gene. The y-axis represents the relative mRNA abundance in NTDs compared with the control. The red column indicates a maternally expressed gene, while the blue column indicates a paternally expressed gene. *: <i>P</i><0.05; **: <i>P</i><0.01; ***: <i>P</i><0.0001 (Student's <i>t</i>-test).</p

Enhanced mRNA levels of <i>IGF2</i> in the spinal cord and brains of human fetuses with NTDs.

<p>(A) In the spinal cord and (B) brains of human fetuses with NTDs, <i>IGF2</i> mRNA levels were dramatically upregulated. *: <i>P</i><0.05; **: <i>P</i><0.01; ***: <i>P</i><0.0001 (Student's <i>t</i>-test).</p

Additional file 3: of Genetic analysis of Wnt/PCP genes in neural tube defects

Figure S2. CELSR2 p.Thr2026Met and DVL3 p.Asp403Asn did not affect the protein subcellular localization. (A) CELSR2 p.Thr2026Met did not affect CELSR2 subcellular localization in HEK293T & MDCKII cells transfected with CELSR2-GFP and CELSR2 (p.Thr2026Met)-GFP expression plasmids. (B) DVL3 p.Asp403Asn did not affect DVL3 subcellular localization in HEK293T. (TIFF 4540 kb

Chromatin status in the promoter region of the <i>IGF2</i> gene is negatively associated with gene expression.

<p>(A) and (D): The genomic profile of genes displayed in UCSC (hg19), the genomic locus of target regions in ChIP assay (black block on the gene schematic) and the CpG island (green block underneath the gene schematic) assayed by the MASSARRAY platform are shown. The capital letter beside the block indicates the corresponding panel below. The number beside the block indicates the genomic location that was targeted. In the ChIP assay results (B and E), the enrichment in the y-axis represents the relative enrichment fold in which the higher enrichment in case or in control is designated as 1. C) and F) indicate the DNA methylation level. Notably, in F, the DNA methylation status of <i>KvDMR1</i> is shown.*: <i>P</i><0.05; **: <i>P</i><0.01 (Student's <i>t</i>-test). TSS: transcription start site.</p

Top networks, related disorders and diseases identified in NTD case, controls and known NTD candidate genes.

<p>Top networks, related disorders and diseases identified in NTD case, controls and known NTD candidate genes.</p