Heat map illustration of the distribution of gene expression among all samples for genes selected by pathways.

<p>A) TLR (Toll-like Receptor) Signaling, B) TCR (T cell receptor) Pathway, Ca²⁺/NFAT Signaling. Red indicates increase and green indicates decrease in relative gene expression for each gene calculated individually across all samples.</p

Gene Expression Barcode for EDS signature genes.

<p>The heat map is derived from individual barcode scores from zero (black) to one (red) with red indicating an increased likelihood that the gene is present in all samples in the experiment from which the tissue specific expression estimate is derived (see McCall et al. for complete description of the barcode algorithm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034951#pone.0034951-McCall1" target="_blank">[21]</a>). As shown, the genes for the EDS signature were overrepresented for three tissues, in particular (cord blood reticulocytes, adult blood reticulocytes, and bone marrow), and their respective number of barcode expression calls are as indicated in the barchart directly below the corresponding areas of the barcode heatmap.</p

Statistically significant differential expression of erythroid CD71+ specific genes.

<p>A) ALAS2 and B) ERAF/AHSP genes in hypertension groups (SSc-PH-ILD, IPAH, and SSc-PAH) versus healthy controls and scleroderma (SSc). The box plots give the mean (horizontal black line), sample values (short blue lines), and 84% confidence interval (CI) (red lines) for each group, (non-overlap of the 84% CIs of two groups is an approximate indicator of significant difference between their means at the 0.05 level of significance). C) Individual ALAS2 and ERAF gene expression microarray results were validated by RT-PCR for high and low EDS patients across all hypertension classes (SSc-PAH, IPAH, SSc-PH-ILD).</p> <p>Further inspection of the EDS gene list also showed the inclusion of genes for both the GATA1 and KLF transcription factors which are both essential for erythroid development [28,29]. A test by gene set analysis of the entire dataset comparing each PH group directly versus the SSc group as the baseline group showed a significant and specific enrichment among genes which contain three different GATA transcription factor binding sites in their upstream promoter regions (TRANSFAC [30]) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034951#pone-0034951-g007" target="_blank">Figure 7A</a>). Interestingly, the genes up-regulated in each of the GATA transcription factor binding site gene sets were mostly non-overlapping either with each other (an average of 59–62% unique genes for each gene list) or with the EDS gene expression signature itself (95% unique non-overlapping genes) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034951#pone-0034951-g007" target="_blank">Figure 7</a>B) indicating that the effects of elevated GATA1 gene expression are both pervasive in the PH groups and are supplemental to the EDS gene expression signature itself. The observation that downstream regulatory events are related directly to EDS elevation (through the up-regulation of the GATA1 transcription factor gene) and are associated with PH groups, taken together with the previously demonstrated strong association of the EDS with reticulocyte maturation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034951#pone-0034951-g005" target="_blank">Figure 5</a>), led us to the identification of this signature as the Erythroid Development Signature (EDS).</p

ALAS2 gene expression positively correlates with increasing disease severity in IPAH patients.

<p>EDS gene expression for 12 IPAH patients for whom hemodynamic measurements were available within four months of the date of blood draw. The clinical parameters of right atrial pressure (RAmean), cardiac index (CI), pulmonary vascular resistance index (PVRI), and pulmonary artery saturation (PA sat) were used. Disease severity in IPAH patients as indicated by increasing RAmean and PVRI are strongly correlated with increasing ALAS2 gene expression and negatively correlated with measures of healthy lung and coronary function such as PA saturation and Cardiac Index (CI).</p

Results from gene set analysis using gene lists derived from the TransFac (gene associated transcription factor binding sites) database.

<p>A). The average changes in gene expression for patients with SSc-PH-ILD, IPAH, and SSc-PAH were tested versus patients with SSc. The p-values associated with the gene set enrichment scores are reported here as −log(p). B). Venn diagram illustrating overlap between the average up-regulated gene expression associated with the presence of 3 distinct GATA transcription factor binding sites. The EDS gene signature is included for comparison.</p

Results from gene set analysis using the EDS as the query gene list. Enrichment scores are as described above.

<p>Results from gene set analysis using the EDS as the query gene list. Enrichment scores are as described above.</p

Heat map of unsupervised clustering (genes only) of 296 genes selected for both high variance (from a set of 500 most variant genes) and also as representative of six major patterns of distinctly correlated genes across the dataset.

<p>These patterns included an Immune Response signature (up- or down-regulated between controls and disease: IR-UR and IR-DR, respectively), the Erythroid Differentiation Signature (EDS) a Platelet specific signature (Pl), and an Immature Neutrophil Signature (INS). The gender specific cluster acts as a positive control, the specific signatures are discussed in detail in the text, particularly that of the EDS.</p

Demographic and clinical characteristics of the patients with PH.

<p>Demographic and clinical characteristics of the patients with PH.</p

Results from gene set analysis of all gene expression data versus gene lists derived from the Mouse Genome Informatics (MGI) database.

<p>A) Gene set enrichment scores were calculated by the PAGE method using row normalized data for all (filtered) genes and samples. The scores were averaged by group and are normalized in the sense that that the average gene expression as scored by PAGE for any one gene set is expressed as the z transformation ((gene set size adjusted) of the average gene expression for that set. Disease specific patterns of gene set enrichment are as shown in the zoomed image. B) Average gene set expression in disease groups versus controls for various blood disorder-specific gene sets (as indicated by arrows in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034951#pone-0034951-g004" target="_blank">Fig. 4</a>A) were tested by Students t test, the results being reported as the negative log (base 10) of the derived p-value (− log(p)).</p