Results of methylation and expression analyses of <i>FAM50B</i> in blood.

<div><p><b>a</b>) <b>Methylation analysis</b>. </p> <p>The figure shows the results of the methylation analyses by deep bisulfite sequencing of two normal controls (NC 4 and NC 15) and the patient (top). Heterozygosity for a SNP in both normal controls allowed to separate the alleles (bottom).</p> <p>Mean methylation levels, SNP allele and number of reads are given below each pattern. Lines represent reads; columns represent CpG dinucleotides; blue squares – unmethylated CpGs; red squares – methylated CpGs; white squares – missing sequence information; * – CpG investigated on the 27k array (CpG 17).</p> <p><b>b</b>) Expression analysis.</p> <p>Expression analysis in four normal controls heterozygous for a SNP (rs6597007 C/G). For each normal control (NC) sequences obtained from peripheral blood DNA and RNA are shown. In the RNA only one allele is present, which indicates monoallelic expression.</p></div

Methylation patterns of <i>ACTN3</i>.

<div><p>The two normal controls investigated display a high variability in methylation levels of adjacent CpGs. The same pattern is visible in the patient in a hypomethylated form.</p> <p>Mean methylation levels, SNP allele and number of reads are given below each pattern. Lines represent reads; columns represent CpG dinucleotides; blue squares – unmethylated CpGs; red squares – methylated CpGs; white squares – missing sequence information; * – CpG investigated on the 27k array (CpG 3).</p></div

Results of methylation and expression analyses of <i>TRPC3</i> in blood.

<div><p><b>a</b>) Methylation patterns of <i>TRPC3</i>.</p> <p>Results of the methylation analyses of 24 CpG dinucleotides obtained by deep bisulfite sequencing. Peripheral blood samples of eight normal controls (NC) and the patient were investigated. The present SNP at CpG 6 indicates the distribution of methylation across the two alleles. As it disrupts the CpG site 6, the white squares refer to the C allele, while filled squares refer to the G allele.</p> <p>Mean methylation levels, SNP allele and number of reads are given below each pattern. Lines represent reads; columns represent CpG dinucleotides; blue squares – unmethylated CpGs; red squares – methylated CpGs; white squares – missing sequence information, at CpG 6 due to a SNP (rs13121031) that disrupts the CpG dinucleotide; * – CpG investigated on the 27k array (CpG 21).</p> <p><b>b</b>) Expression patterns of <i>TRPC3</i> in blood.</p> <p>Expression analyses in three normal controls heterozygous for a SNP (rs11732666 G/A). For each normal control (NC) sequences obtained from peripheral blood DNA and RNA are shown. Expression varies from biallelic to skewed. </p></div

Results of the pseudoproband approach.

<div><p>The diagram shows the number of CpGs in all investigated individuals passing the threshold of delta β ≥ 0.3 when assigned as a pseudoproband. All imprinted genes were excluded. </p> <p>a - t – healthy controls; M – mother of the patient; F – father of the patient; average – average number of CpGs showing a deviation in normal controls and the parents.</p></div

Die Rolle der Antileukoproteinase SLPI bei der Infektion mit HPV bei Kopf-Hals-Karzinomen

<div><p>Treatment with recombinant human growth hormone (rhGH) has been consistently reported to induce transcriptional changes in various human tissues including peripheral blood. For other hormones it has been shown that the induction of such transcriptional effects is conferred or at least accompanied by DNA-methylation changes. To analyse effects of short term rhGH treatment on the DNA-methylome we investigated a total of 24 patients at baseline and after 4-day rhGH stimulation. We performed array-based DNA-methylation profiling of paired peripheral blood mononuclear cell samples followed by targeted validation using bisulfite pyrosequencing. Unsupervised analysis of DNA-methylation in this short-term treated cohort revealed clustering according to individuals rather than treatment. Supervised analysis identified 239 CpGs as significantly differentially methylated between baseline and rhGH-stimulated samples (p<0.0001, unadjusted paired t-test), which nevertheless did not retain significance after adjustment for multiple testing. An individualized evaluation strategy led to the identification of 2350 CpG and 3 CpH sites showing methylation differences of at least 10% in more than 2 of the 24 analyzed sample pairs. To investigate the long term effects of rhGH treatment on the DNA-methylome, we analyzed peripheral blood cells from an independent cohort of 36 rhGH treated children born small for gestational age (SGA) as compared to 18 untreated controls. Median treatment interval was 33 months. In line with the groupwise comparison in the short-term treated cohort no differentially methylated targets reached the level of significance in the long-term treated cohort. We identified marked intra-individual responses of DNA-methylation to short-term rhGH treatment. These responses seem to be predominately associated with immunologic functions and show considerable inter-individual heterogeneity. The latter is likely the cause for the lack of a rhGH induced homogeneous DNA-methylation signature after short- and long-term treatment, which nevertheless is well in line with generally assumed safety of rhGH treatment.</p></div

DNA methylation differs between AC and CVS samples.

<p>(A) Principal component analysis (PCA) of 22,234 CpG loci which passed quality tests and entered the analysis. Unsupervised analysis separated AC (green spheres) from CVS samples (red spheres). (B) Differential methylation analysis to separate AC from CVS samples (t-test, FDR<1×10⁻¹⁵). (C) Hierarchic cluster analysis of the 2418 CpG loci shown in (B). The upper panel on top of the heatmap indicates the sample type (green: AC, red:CVS) while the second panel indicates the fetuses’ sex (pink: female, cyan: male). Green color in the heatmap indicates low DNA-methylation, black intermediate and red high DNA-methylation values. Samples with known chromosomal aberrations and samples from ICSI were excluded. AC and CVS differ significantly in their DNA methylation pattern.</p

Increase of IGF1 concentration upon rhGH treatment in the IGFGT.

<p>Scatter plot of the IGF1 concentrations (ng/ml) at baseline and rhGH stimulated blood sampling among the short-term rhGH treatment study cohort. The two measurements per sample are connected. Sample numbering corresponds to the sample identifier in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120463#pone.0120463.t001" target="_blank">Table 1</a>. A significant increase in IGF1 concentrations after rhGH treatment is observed (paired t-test, p = 1.93 x 10–6).</p

DNA-methylation results in the short-term rhGH treatment study cohort using an individualised evaluation approach.

<p>Differences of avg.beta values between stimulated and baseline samples (delta beta) of each pair were calculated. Depicted are those loci, in which at least 3/24 analysed sample pairs showed absolute delta beta values below -0.1 or above 0.1 (roughly corresponding to a difference of DNA-methylation of +/- 10% between each analysed sample pair) and in which at least 3 CpGs were affected per gene (n = 259). Red: delta beta values above 0.2, pale red: delta beta values between 0.1 and 0.2. Blue: delta beta values below -0.2, pale blue: delta beta values between -0.1 and -0.2. Samples are sorted according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120463#pone.0120463.t001" target="_blank">Table 1</a> with P1 at the left margin and P26 at the right margin. Those loci used for bisulfite pyrosequencing validation are zoomed out and shown at the right side of the figure.</p

DNA methylation patterns in CVS samples with chromosomal imbalances.

<p>PCA (A) and Hierarchic cluster analysis (B) of DNA methylation values of 464 CpG loci differentially methylated between chorionic villi biopsy samples without chromosomal imbalances (red spheres or red squares, respectively) and samples with trisomy 21 (47,XY+21; blue spheres or blue squares, respectively) (FDR<0.01). While in cases with trisomy 21 numerous epigenetic alterations could be identified, in cases with trisomy 18 (47,XX+21/47,XY+21, yellow spheres or yellow squares) only 15 differentially methylated loci have been found (C and D). Green color in the heatmap indicates low DNA-methylation, black intermediate and red high DNA-methylation values.</p

Hierarchic cluster analysis of DNA methylation values of 767 X-chromosomal CpG loci present on the array.

<p>The upper panel on top of the heatmap indicates the sample type (green: AC, red: CVS) while the second panel indicates the fetuses’ sex (pink: female, cyan: male). Samples with known chromosomal aberrations and samples from ICSI were excluded. Three CVS samples from male fetuses show a DNA methylation pattern similar to female samples (arrow). A blue bar at the right site indicates genes methylated specifically in male fetuses, an orange bar genes methylated in female samples and a black bar genes with tissue- specific DNA methylation.</p