Absolute count of immune cells in children with Down syndrome (DS) compared with controls.

a<p> Data are expressed as mean ± standard deviation, and two-tailed t test was used for comparisons.</p>b<p> Data is expressed as median (interquartile range), and Mann-Whitney test was used.</p><p>Absolute count of immune cells in children with Down syndrome (DS) compared with controls.</p

The maternally derived, imprinted 5^mCpG marks at the <i>WRB</i> CGI-2 DMR do not dictate a paternal monoallelic expression in blood.

<p>Qualitative SNuPE allele-specific profiling of the <i>WRB</i> 3´-UTR rs1060180, rs13230 and rs60490159 SNPs in a disomic, informative nuclear family (DNA). The assay reveals a pattern consistent with biallelic transcriptional expression (cDNA) in the child, who is heterozygous for all three SNP variants.</p

Common combinatorial histone modification expression patterns around the <i>WRB</i> CGI-2 DMRs.

<p>Graphical representation of the confluence of activating and repressive epigenetics histone modification marks for the known imprinted <i>MEST</i> gene (<b>A</b>) and the candidate imprinted <i>WRB</i> gene (<b>B</b>). Shown are the 17-histone modification activation backbone module and the 5-histone modification repressive module found in human CD4+ T cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154108#pone.0154108.ref054" target="_blank">54</a>]. Highlighted in light blue is the DMR in each gene. Composite of screenshots of the dataset viewed at the UCSC Genome Browser hg18 (<a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu</a>).</p

The maternally derived, imprinted 5^mCpG marks at the <i>WRB</i> CGI-2 DMR do not dictate a paternal monoallelic expression in a human embryonic stem cell line.

<p>Allele-specific transcriptional profiling of the <i>WRB</i> 3´-UTR rs1060180 SNP in the informative hESC HUES 1 sample (DNA; upper panel) reveals a pattern consistent with biallelic expression (cDNA; lower panel). In contrast, for the known paternally imprinted <i>H19</i> and <i>ATP10A</i> genes, the expression profiles for the informative rs2839702 and rs2076743 SNPs, respectively, are monoallelic.</p

The <i>WRB</i> CGI-2 DMR contains a primate-conserved cluster array of a DNA motif.

<p>(<b>A</b>) Molecular phylogenetic relationship of eight <i>WRB</i> CGI-2 DNA primate reference sequences inferred using multiple sequence alignment by CLUSTALW. (<b>B</b>) Sequence logo of the primate-conserved [AGGYGBYSYAGGACT] DNA repeat unit that occurs at the <i>WRB</i> CGI-2. (<b>C</b>) Physical map of the primate-conserved [AGGYGBYSYAGGACT] DNA repeat unit array.</p

5^mCpG statuses at the <i>WRB</i> CGI-2 DMR in a complete androgenetic mole and a human embryonic stem cell line.

<p>A consistent unmethylated pattern of CpG sites at the <i>WRB</i> CGI-2 DMR revealed in a sample of an androgenetic complete hydatidiform mole (<b>A</b>) contrast with the hypermethylated pattern observed in the representative HUES 3 embryonic cell line (<b>B</b>). Electropherograms of the amplimers (see details of the assay in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154108#pone.0154108.g002" target="_blank">Fig 2</a>) generated from either undigested DNA or <i>Hha</i>I-digested DNA. The numbers in the upper boxes correspond to the amplimer lengths in base pairs while those in the lower boxes refer to the areas under the peak of the amplimer.</p

Maternal-of-origin-dependent imprinted methylation marks at the <i>WRB</i> CGI-2 DMR.

<p>5^mCpG-sensitive restriction endonuclease sites at the <i>WRB</i> CGI-2 DMR are differentially methylated on the maternal allele <i>versus</i> the paternal allele in a manner consistent with imprinting. Electropherograms of the genotype profiles in a control nuclear family, informative for the rs2244352 (C>A) SNP neighboring the <i>WRB</i> CGI-2 DMR. In the child, the maternal-derived C allele is 100% resistant to <i>Hha</i>I digestion (fully methylated), whereas the paternal-derived A allele is 100% susceptible to <i>Hha</i>I digestion (i.e., unmethylated). The parental allele-specific methylation statuses were validated using the <i>McrBC</i> restriction endonuclease that cleaves methylated DNA and, therefore, the unmethylated paternal allele, but not the maternally methylated allele, remains undigested.</p

Experimental validation of the 5^mCpG statuses at the <i>WRB</i> CGIs.

<p>(<b>A</b>) A consistent hemimethylated pattern at the <i>WRB</i> CGI-2 revealed in a representative control disomic DNA sample (blood) using the Hha<i>I</i> methylation-sensitive restriction enzyme-based PCR triplex assay developed in this study. Electropherograms of the amplimers generated from either undigested genomic DNA (upper panel) or DNA digested with <i>Hha</i>I (lower panel) genotyped via quantitative fluorescent PCR. The positive amplimer refers to a <i>locus</i> in the <i>ESCO2</i> gene with constitutively hypomethylated CpG dinucleotides at the target restriction enzyme sites (100% susceptible to <i>Hha</i>I digestion). The negative amplimer refers to a <i>WRB</i> region that lacks <i>Hha</i>I sites, and is, therefore, refractory to enzymatic digestion. The numbers in the upper boxes correspond to the amplimer lengths in base pairs while those in the lower boxes refer to the areas under the peak of the amplimer. In this representative DNA sample, the ratio of 5^mCpG sites at the <i>WRB</i> CGI-2 was 50.6%. In contrast, the assay revealed a consistent unmethylated pattern of CpG sites at the <i>WRB</i> CGI-1 (<b>B</b>) and <i>WRB</i> CGI-3 (<b>C</b>).</p

The maternal (oocyte)-derived allele methylation at the <i>WRB</i> CGI-2 distinguishes the parental origin of chromosome 21 nondisjunction events in Down syndrome probands.

<p>The proportion of <i>Hha</i>I-resistant 5^mCpG sites at the <i>WRB</i> CGI-2 DMR in trisomic samples with a maternal origin (MT21) of the nondisjoined chromosome 21 is consistently and statistically higher than in trisomic samples with a paternally (PT21) derived extra copy of chromosome 21. In genomic DNA from an androgenetic hydatidiform mole (ACHM), the <i>WRB</i> CGI-2 is unmethylated, whereas it is hypermethylated in hESCs. In control disomic samples (N21) the <i>locus</i> is partially methylated, consistent with a hemimethylated state. All differences were statistically significant.</p

The methylation statuses at the CpG islands of the <i>WRB</i> gene in methylome public datasets.

<p>Chromosomal, physical map positions and sequence features of the reference <i>WRB</i> gene <i>locus</i> showing the methylation profiles across the region, with an emphasis on the five annotated CpG islands (CGI-1 to CGI-5). The features depicted are from custom and public tracks for (from top to bottom) the exon organization of the principal (ENST00000333781.8) and alternative (ENST00000380708.4) splice <i>WRB</i> isoforms (variants 1 and 2 in dark blue) (UCSC Genes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154108#pone.0154108.ref036" target="_blank">36</a>]), the species-conserved principal transcript (ENST00000333781 in pink) (APPRIS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154108#pone.0154108.ref075" target="_blank">75</a>]), the Ensembl Regulatory Build CTCF and POL2 activity and predicted promoters [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154108#pone.0154108.ref076" target="_blank">76</a>], CpG islands, CpG sites, and regulation and methylome studies indicated in the Materials and Methods section. The CGI-2 is located in the differentially methylated region (DMR) reported by Court and collaborators [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154108#pone.0154108.ref024" target="_blank">24</a>] and Docherty and collaborators [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154108#pone.0154108.ref025" target="_blank">25</a>] (depicted in red in the custom track named Court/Docherty DMR), from which <i>WRB</i> was classified originally as a novel candidate, maternally imprinted gene (i.e., paternal-origin allele expressed). The custom tracks containing the sperm and oocyte DNA methylation signals correspond to the supplementary data reported by Okae <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154108#pone.0154108.ref046" target="_blank">46</a>]. Screenshot generated using the UCSC Genome Browser hg19 (<a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu</a>).</p