Erythroid-Specific Transcriptional Changes in PBMCs from Pulmonary Hypertension Patients

Gene expression profiling of peripheral blood mononuclear cells (PBMCs) is a powerful tool for the identification of surrogate markers involved in disease processes. The hypothesis tested in this study was that chronic exposure of PBMCs to a hypertensive environment in remodeled pulmonary vessels would be reflected by specific transcriptional changes in these cells.The transcript profiles of PBMCs from 30 idiopathic pulmonary arterial hypertension patients (IPAH), 19 patients with systemic sclerosis without pulmonary hypertension (SSc), 42 scleroderma-associated pulmonary arterial hypertensio patients (SSc-PAH), and 8 patients with SSc complicated by interstitial lung disease and pulmonary hypertension (SSc-PH-ILD) were compared to the gene expression profiles of PBMCs from 41 healthy individuals. Multiple gene expression signatures were identified which could distinguish various disease groups from controls. One of these signatures, specific for erythrocyte maturation, is enriched specifically in patients with PH. This association was validated in multiple published datasets. The erythropoiesis signature was strongly correlated with hemodynamic measures of increasing disease severity in IPAH patients. No significant correlation of the same type was noted for SSc-PAH patients, this despite a clear signature enrichment within this group overall. These findings suggest an association of the erythropoiesis signature in PBMCs from patients with PH with a variable presentation among different subtypes of disease.In PH, the expansion of immature red blood cell precursors may constitute a response to the increasingly hypoxic conditions prevalent in this syndrome. A correlation of this erythrocyte signature with more severe hypertension cases may provide an important biomarker of disease progression

Understanding the biology of inflammatory breast cancer cutaneous metastasis and the role of TGFβ

van Golen, Kenneth L.Inflammatory breast cancer (IBC) is a highly aggressive form of locally advanced breast cancer with unique molecular and phenotypic properties (Dawood, Merajver et al. 2011, Dawood and Valero 2012, Joglekar and van Golen 2012). Cutaneous metastases from internal cancers are relatively rare, occurring at a rate of 0.7-9.0% (Martin 1997). IBC cutaneous metastasis is associated with chest wall recurrences, significantly decreasing the quality of life and survival (Cristofanilli, Valero et al. 2007). Although significantly different in many aspects, IBC and melanoma share a number of similarities in disease presentation and progression. Both spread via dermal lymphatics, form intralymphatic emboli and have a propensity to form cutaneous metastases (Fidler 1990, Leiter, Meier et al. 2004, Rose, Christos et al. 2011). Thus, new leads for studying cutaneous metastasis can be gathered from the melanoma literature. In melanoma, there were several studies done on radiation and TGFβ to demonstrate the role of TGFβ on the etiology of melanoma cutaneous metastasis (Schmid, Itin et al. 1995, Perrot, Javelaud et al. 2013). There were no studies done to date to understand the biology of inflammatory breast cancer cutaneous metastasis in relation to radiation and the role of TGFβ. Here my doctoral project is primarily focusing on the influence of radiation in IBC cutaneous metastasis and the role of TGFβ. I radiated normal human fibroblast cells with different doses of radiation and used the conditioned media to see the invasiveness of the IBC cells (KPL4 and SUM149) and compared them with the conventional breast cancer cells (MDA-MB-231). I observed that the IBC cell invasion is significantly higher with higher doses of radiation. Also there is higher expression of TGFβ-2 with higher (5 Gy) dose of radiation. I also used RNA sequencing to identify the molecular signature profile of IBC and non-IBC cells which may lead to the therapeutic treatment of IBC cutaneous metastasis.University of Delaware, Department of Medical Laboratory SciencesPh.D

Replicated methylation changes associated with eczema herpeticum and allergic response.

BackgroundAlthough epigenetic mechanisms are important risk factors for allergic disease, few studies have evaluated DNA methylation differences associated with atopic dermatitis (AD), and none has focused on AD with eczema herpeticum (ADEH+). We will determine how methylation varies in AD individuals with/without EH and associated traits. We modeled differences in genome-wide DNA methylation in whole blood cells from 90 ADEH+, 83 ADEH-, and 84 non-atopic, healthy control subjects, replicating in 36 ADEH+, 53 ADEH-, and 55 non-atopic healthy control subjects. We adjusted for cell-type composition in our models and used genome-wide and candidate-gene approaches.ResultsWe replicated one CpG which was significantly differentially methylated by severity, with suggestive replication at four others showing differential methylation by phenotype or severity. Not adjusting for eosinophil content, we identified 490 significantly differentially methylated CpGs (ADEH+ vs healthy controls, genome-wide). Many of these associated with severity measures, especially eosinophil count (431/490 sites).ConclusionsWe identified a CpG in IL4 associated with serum tIgE levels, supporting a role for Th2 immune mediating mechanisms in AD. Changes in eosinophil level, a measure of disease severity, are associated with methylation changes, providing a potential mechanism for phenotypic changes in immune response-related traits

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

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

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

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

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