Additional file 2 of CC-PROMISE effectively integrates two forms of molecular data with multiple biologically related endpoints

This PDF file provides plots of the probability of significance as a function of the p-value threshold for all seven analysis methods in each of the 500 simulation settings. The plots have a similar interpretation as those in Fig. 2. (PDF 1263 kb

Additional file 4 of CC-PROMISE effectively integrates two forms of molecular data with multiple biologically related endpoints

This PDF file provides technical details regarding the CC-PROMISE analysis performed for the example application involving pediatric acute myeloid leukemia described in subsection “Acute myeloid leukemia example”. Details include description of the specific association statistics used for each molecular data set and each endpoint, the coefficients used in the analysis, and the number of permutations performed. (PDF 266 kb

Analysis of MDM2 and MDM4 Single Nucleotide Polymorphisms, mRNA Splicing and Protein Expression in Retinoblastoma

<div><p>Retinoblastoma is a childhood cancer of the developing retina that begins in utero and is diagnosed in the first years of life. Biallelic <em>RB1</em> gene inactivation is the initiating genetic lesion in retinoblastoma. The p53 gene is intact in human retinoblastoma but the pathway is believed to be suppressed by increased expression of MDM4 (MDMX) and MDM2. Here we quantify the expression of MDM4 and MDM2 mRNA and protein in human fetal retinae, primary retinoblastomas, retinoblastoma cell lines and several independent orthotopic retinoblastoma xenografts. We found that MDM4 is the major p53 antagonist expressed in retinoblastoma and in the developing human retina. We also discovered that MDM4 protein steady state levels are much higher in retinoblastoma than in human fetal retinae. This increase would not have been predicted based on the mRNA levels. We explored several possible post-transcriptional mechanisms that may contribute to the elevated levels of MDM4 protein. A proportion of MDM4 transcripts are alternatively spliced to produce protein products that are reported to be more stable and oncogenic. We also discovered that a microRNA predicted to target MDM4 (miR191) was downregulated in retinoblastoma relative to human fetal retinae and a subset of samples had somatic mutations that eliminated the miR-191 binding site in the MDM4 mRNA. Taken together, these data suggest that post-transcriptional mechanisms may contribute to stabilization of the MDM4 protein in retinoblastoma.</p> </div

Expression of MDM4 in retinoblastoma.

<p><b>A</b>) Structure of the genomic organization of the 11 exons of human <i>MDM4</i>. The exons are to scale as shown but the introns are not to scale. (<b>B</b>) Schematic of the spliced <i>MDM4</i> mRNA that produces the full-length MDM4 protein. The location and identification of Affymetrix gene expression array probesets are shown in red and the location of the real time RT-PCR probe/primer set is shown in blue. (<b>C</b>) Boxplots of the Log₂ expression of each of the 5 probesets that uniquely mapped to <i>MDM4</i> for fetal retina, cell lines, xenografts and 52 primary human retinoblastomas. (<b>D</b>) Real time RT-PCR for MDM2 using Taqman probes as shown in (B) for fetal retina at gestational stage week 20 (FW20), control cell lines (NB1691 and U20S), retinoblastoma cell lines (Y79, Weri1, RB355), primary tumors (SJ33, SJ43, SJ45), and retinoblastoma xenografts (SJ39-X, SJ41-X, SJ42-X and MSK176). Values were normalized to the positive control (U2OS). (<b>E</b>) The same data as shown in (D) but normalized to fetal retina. Each bar is the mean and standard deviation of duplicate experiments. (<b>F</b>) The SAGE data from developing mouse retina shows <i>Mdm4</i> is expressed at significant levels in the developing mouse retina. The gray shaded box represents the limit of detection by SAGE (<2 normalized tags per sample). <i>Gapdh</i> and <i>Gpi1</i> are plotted as ubiquitiously expressed internal controls. (<b>G</b>) Immunoblot of MDM4 (green) protein in a subset of the samples analyzed by real time RT-PCR. Gapdh (red) was used as an internal reference for gel loading and the normalized values are presented below the black and white presentation of the blot for MDM4. The antibody was verified for specificity using a blocking peptide (not shown). Recombinant full length Flag-MDM4 protein was included as a positive control. Multiple bands were detected for MDM4 and were quantitated (below the black and white picture of the blot) and normalized to GAPDH and relative to U2OS cell line.</p

Expression of MDM2 in retinoblastoma.

<p><b>A</b>) Structure of the genomic organization of the 11 exons of human <i>MDM2</i>. The exons are to scale as shown, but the introns are not to scale. <b>B</b>) Schematic of the spliced <i>MDM2</i> mRNA that produces the full-length MDM2 protein. The location and identification of Affymetrix gene expression array probesets are shown in red and the location of the real time RT-PCR probe/primer set is shown in blue. (<b>C</b>) Boxplots of the Log₂ expression of each of the 6 probesets that uniquely mapped to <i>MDM2</i> for fetal retina, cell lines, xenografts and 52 primary human retinoblastomas. In general, genes expressed below Log₂ of 7–8 cannot be detected by SAGE and are below the limit of reliable detection for retinal samples. (<b>D</b>) Real time RT-PCR for <i>MDM2</i> using Taqman probes as shown in (B) for fetal retina at gestational stage week 20 (FW20), a control (NB1691) that expresses high levels of MDM2, and a control (U2OS) that expresses high levels of MDM4, retinoblastoma cell lines (Y79, Weri1, RB355), primary tumors (SJ33, SJ43, SJ45), and retinoblastoma xenografts (SJ39-X, SJ41-X, SJ42-X and MSK176). Values were normalized to the positive control (NB1691). (<b>E</b>) The same data as shown in (D) but normalized to fetal retina. Each bar is the mean and standard deviation of duplicate experiments. (<b>F</b>) The SAGE data from developing mouse retina shows <i>Mdm2</i> is not expressed at significant levels in the developing mouse retina. The gray shaded box represents the limit of detection by SAGE (<2 normalized tags per sample). <i>Gapdh</i> and <i>Gpi1</i> are plotted as ubiquitiously expressed internal controls. (<b>G</b>) Immunoblot of MDM2 (green) protein in a subset of the samples analyzed by real time RT-PCR. Gapdh (red) was used as an internal reference for gel loading and the normalized values are presented below the black and white presentation of the blot for MDM2.</p

Analysis of transcript variants of MDM4 in retinoblastoma.

<p><b>A, B</b>) Genomic organization of MDM4 showing two previously identified splice variants for this gene and the corresponding mRNA. MDM4-S results from skipping of exon 6 and MDM4-A results from skipping of exon 9 (dark gray shading). (<b>C</b>) Domain structure of the full length MDM4 protein with known protein modification sites (phosphorylation (P) and sumoylation (S)) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042739#pone.0042739-Mancini1" target="_blank">[18]</a>). The predicted size is 55 kDa with an observed size on SDS-PAGE of ∼75 kDa. (<b>D</b>) The loss of exon 6 in MDM4-S results in a frameshift after residue 114 and a translational termination after residue 140 (diagonal line). The predicted size of MDM4-S is 15 kDa and the observed size on SDS-PAGE is 27 kDa <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042739#pone.0042739-Mancini1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042739#pone.0042739-Rallapalli3" target="_blank">[27]</a>. (<b>E</b>) The loss of exon 9 in MDM4-A results in an in frame fusion that is missing 50 residues spanning a portion of the acidic domain. (<b>F</b>) Representative reads from transcriptome analysis showing the loss of exon 6 and exon 9 in the retinoblastoma orthotopic xenografts. These alternative spliced transcripts were verified by PCR (<b>H</b>) and Sanger sequencing (<b>I</b>). (<b>G</b>) Representative reads from transcriptome analysis of MDM2 showing very little detection for alternative spliced transcripts.</p

Analysis of miR-191 in retinoblastoma.

<p><b>A</b>) Boxplot of the Log₂ expression for hsa-mir-191 probeset in 6 fetal retina and 16 primary human retinoblastomas showing significantly lower expression. (<b>B</b>) MicroRNA real time-RT-PCR for hsa-mir-191 in fetal retina at gestational week 20, retinoblastoma cell lines (Rb355, Y79, and Weri1), retinoblastoma primary tumors, and orthotopic xenografts. Values are normalized to fetal retina. (<b>C</b>) Scatter plots of the Log₂ expression values for hsa-mir-191 and 4 of the representative 44 mir-191 target genes in fetal retina and retinoblastoma primary tumors. Affymetrix probeset in parenthesis. (<b>D</b>) Scatter plot of the Log₂ expression values for hsa-mir-191 and MDM4 (Affymetrix probeset 236814_at) in retinoblastoma primary tumors. Circled are primary tumors with a C/A genotype.</p