Clinical information for the 20 patients with acute lymphoblastic leukemia and 13 controls included in the study.

<p>BCP indicates B-cell precursor ALL; T-ALL, T-cell ALL; HeH, high hyperdiploidy; amp(21), amplification of chr 21; HR, high risk; SR, standard risk; IR, intermediate risk; NA, not available.</p>a<p>White blood cell count at diagnosis (10⁹/L).</p>b<p>The NOPHO ALL 2000 protocol was used.</p>c<p>DNA from was available from bone marrow taken from the patients on day 29,50, and/or 106 after the initiation of therapy, all patients were in morphological remission with less than 5% leukemic blasts.</p

Correlation matrix and variability of the methylation levels measured at 1,320 CpG sites across the 63 samples included in the study.

<p>(A) Each individual sample is indicated by a black line on the axes. The methylation levels in the samples taken at remission during induction therapy at day 29 and during consolidation therapy at days 50 and 106 are highly correlated with the methylation levels in the non-leukemic samples (median Pearson's correlation coefficient (<i>R</i>) = 0.96), while the diagnostic ALL samples are less similar both to each other and to the samples taken after treatment, and to the non-leukemic samples (median <i>R</i> = 0.83). The scale for the correlation coefficients is shown to the right of the matrix. The red color indicates higher correlation (greater similarity), while the light yellow indicates less correlation (less similarity). (B) Histograms of the standard deviations (SD) for the methylation levels measured for 1,320 CpG sites across 20 ALL samples (blue) and across the combined 33 remission samples and 13 non-leukemic controls (red). SD bins are shown on the horizontal axis. The vertical bars show the proportion of observations in each SD bin. The CpG sites show greater variability in the ALL samples than in the remission samples and non-leukemic controls (Wilcoxon Rank-Sum P<0.001).</p

Differential methylation in ALL cells.

<p>(A) Heatmap of the methylation profiles of the 28 CpG sites that are differentially methylated between the diagnostic ALL samples, bone marrow cells at remission and non-leukemic bone marrow cells. The ALL samples (orange) and bone marrow cells during remission (blue) form two distinct groups. Thirteen bone marrow cell samples from non-leukemic controls (purple) cluster among the samples collected during remission. The scale for the methylation β-values is shown below the heatmap. The elongated heights of the dendrogram branches between the ALL samples compared to the normal samples illustrate the increased variability in the ALL samples for the 28 CpG sites. Graphs showing the differences in methylation level between CpG sites in the (B) <i>WDR35</i> and (C) <i>FXYD2</i> genes at the time of diagnosis (left vertical axis) and during remission (right vertical axis). The data points for each paired sample are connected with a red line for B-cell precursor (BCP) samples and with a blue line for T-ALL samples. The corresponding CpG methylation levels in 13 non-leukemic control samples are shown as black horizontal lines to the right of the graphs. The CpG site at chr2:20,052,748 in the <i>WDR35</i> gene (B) was hypermethylated in diagnostic ALL samples and hypomethylated at remission and in non-leukemic controls, while the CpG site at chr11:7,203,745 in the <i>FXYD2</i> gene (C) displayed the opposite pattern. The BCP and T-ALL samples display the same pattern of methylation difference in these two genes.</p

Correlation between the methylation levels (β-values) of two CpG sites located in the <i>COL6A2</i>, <i>EYA4</i>, <i>FXYD2</i> and <i>MYO3A</i> genes.

<p>The Pearson's correlation coefficients (<i>R</i>) across the 20 acute lymphoblastic leukemia (ALL) samples taken at ALL diagnosis (green) and the 20 matched bone marrow samples taken at remission (blue) for the four genes are shown in panels A–D. The positions of the CpG sites for which the β-values are plotted are indicated on the axes in each panel (Human Genome Build 36). The inter-individual variation between the pairs of CpG sites in the remission cells is consistently lower than between the ALL cells, which speaks against the variation in ALL cells arising because of methodological factors.</p

Comparison of total expression levels of regions annotated to intergenic lncRNAs, exons, introns, and intergenic regions.

<p>The horizontal axes in the panels show bins of fluorescence signals from the genotyping data, summed for both alleles to give a measure for total expression. The average expression levels of annotated transcripts were 4900 fluorescence units in exons, 2100 in introns, 590 in intergenic regions, compared to 3300 in the intergenic lncRNA regions that were used in the ASE analysis. The vertical axes show the number of observations in each bin.</p

Illustration of a region with a SNP from genome wide association studies (GWAS) which is associated with ASE of lncRNAs.

<p>The tracks are from top to bottom in each panel: Horizontal red bars represent lncRNA transcript windows (with genomic coordinates) used for determination of ASE levels; grey lines show p-values for the association of GWAS SNPs with ASE levels in the transcript window; a grey line overlayed with a red dotted line indicates that a <i>cis</i>-rSNP overlaps with the reported SNP in the GWAS catalog; red vertical lines are median ASE-levels for each SNP.</p

SNPs associated with allele-specific expression of lncRNA windows with published trait- or disease-associations from genome-wide association studies.

1<p>The distance to the lncRNA region is 0 if the SNP is located within the region and the smallest distance otherwise,</p>2<p>Slope is given in absolute numbers,</p>3<p>Listed are all cis-rSNPs that are also found in the GWAS catalog together with the associated lncRNA,</p>4<p>The trait is taken from the GWAS catalog,</p>5<p>Within 2.5 kb,</p>6<p>rs6663565 as proxy,</p>7<p>rs2303393 as proxy,</p>8<p>rs6922111 as proxy,</p>9<p>rs7739434 as proxy,</p>10<p>rs13214831 as proxy,</p>11<p>rs1153862 as proxy,</p>12<p>rs12442557 as proxy.</p

Manhattan plot.

<p>Manhattan plot with the p-values from ASE association tests between SNPs and lncRNAs on the vertical axis and the genomic lncRNA regions analysed in the study on the horizontal axis. The p-value cut-off of 10⁻⁶ is shown as a grey line.</p

Co-expressed lncRNA regions and protein-coding genes.

1<p>All 52 significantly co-expressed lncRNA and protein coding genes with a lncRNA associated <i>cis</i>-rSNP are listed.</p>2<p>Multiple testing correction using FDR of 10%,</p>3<p>The p-value cut-off for significant ASE is 10⁻⁶,</p>4<p>ASE p-value for a Refseq gene is shown for all regions that are co-expressed with a lncRNA and have an overlapping ASE analysis window, NA otherwise.</p

The ability of ASE and GTE analysis to detect significantly associated rSNPs at different MAF.

<p>Fractions of rSNPs are shown for different minor allele frequencies (MAF) with significant association signals according to a Bonferroni-corrected p-value of 0.05. Each data point underlying the curves represents the fraction of significant associations within a 1% MAF bin. Sliding 5% MAF window averages are plotted for different sample sizes analyzed by ASE and GTE. Both methods detect a lower fraction of low frequency rSNPs, compared to the fraction of all the SNPs at the same frequency (black line). The ASE method detects a higher fraction of the SNPs (solid lines) with a MAF <15% than GTE (dashed lines) regardless of sample size except for the largest GTE sample set.</p