scholarly journals Characterization of Whole Blood Gene Expression Profiles as a Sequel to Globin mRNA Reduction in Patients with Sickle Cell Disease

PLoS ONE ◽  
2009 ◽  
Vol 4 (8) ◽  
pp. e6484 ◽  
Author(s):  
Nalini Raghavachari ◽  
Xiuli Xu ◽  
Peter J. Munson ◽  
Mark T. Gladwin
Keyword(s):  
Gene Expression ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Whole Blood ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Cell Disease ◽  
Globin Mrna ◽  
Blood Gene Expression

Increased Inflammatory State and Metabolic Activation in Neutrophils from Patients with Sickle Cell Disease: Comparison of Neutrophil Gene Expression Profiles with Controls.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3571-3571
Author(s):  
Sule Bakanay ◽  
Ferdane Kutlar ◽  
Joshi Ratanmani ◽  
Betsy Clair ◽  
Leigh Wells ◽  
...  
Keyword(s):  
Gene Expression ◽  
Steady State ◽  
Sickle Cell Disease ◽  
Disease Severity ◽  
Sickle Cell ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Cell Disease ◽  
Expression Of Genes ◽  
Inflammatory State

Abstract Chronic inflammation is a well-established feature of sickle cell disease (SCD) even at steady state, and the degree of inflammation tends to correlate with disease severity. Elevated neutrophil count, as a reflection of the overall inflammatory state, has emerged as an indicator of poor prognosis and has been associated with adverse outcomes including stroke and early mortality. To further delineate the role of neutrophils in the pathogenesis of various complications and in overall disease severity in SCD, we analyzed the gene expression profiles of neutrophils from 5 patients with "severe" disease (>3 vaso-occlusive episodes [VOE] per year), 5 patients with "mild" disease (<3 VOE/year) and compared these to each other and to the gene expression profiles of neutrophils from 5 age and sex matched, healthy, non-sickle cell, African-American individuals. Granulocytes were separated from freshly collected venous blood using Histopaque (Sigma diagnostic) density gradient separation. Total RNA was extracted immediately after cell separation by using Rneasy Mini Kit (Qiagen). 2 micrograms of total RNA was converted to double stranded cDNA (ds-cDNA) by using SuperScript Choice System (Invitrogen). In vitro transcription was performed on the ds-cDNA using Enzo RNA transcript labelling kit. After the fragmentation, labeled RNA was hybridized to a set of oligonucleotide arrays (HG U133A, Affymetrix, Santa Clara, CA) and the data was analysed with the Microarray suite 5.0 software (Affymetrix). Out of the differentially expressed genes (314 genes for severe vs. control, 718 genes for mild vs control), those with greater than two fold expression were analysed with the geneMAPP software for localization into biological pathways. In general, a larger number of genes were differentially expressed between "mild" patients vs. control, compared to that between "severe" vs "mild" patients. Genes related to cellular proliferation, growth and maintenance, DNA repair, DNA replication, and cell cycle progression were expressed at significantly higher levels in SCD patients compared to controls. The most impressive finding was the significantly higher expression of genes leading to NFkB activation and inhibition of apoptosis: IAP-1 (increased 6.7 fold and 4.7 fold in mild and severe patients respectively), IkB (decreased 0.14 fold and 0.3 fold), Apaf-1 (decreased 0.4 fold in mild), and c-jun (decreased 0.4 fold in severe); Traf-2 (TNF receptor associated factor-2; increased 3.5 fold and 2 fold); genes in the MAPK signalling pathway: ERK-2 (increased 3.5 fold and 2-fold), MAP2K3 (increased 3.5 fold and 2 fold). These data show that neutrophils in SCD patients are activated with higher expression of genes in the TNF, MAPK, and NFkB pathways consistent with an inflammatory state. Delayed or inhibited apoptosis of neutrophils further maintains this inflammatory state even during the so-called "steady state" of the disease. We conclude that the analyses of gene expression in neutrophils can be a useful tool in identifying pathways and genes that distinguish SCD patients from controls and in differentiating mild and severe phenotypes.

scholarly journals Blood mononuclear cell gene expression profiles characterize the oxidant, hemolytic, and inflammatory stress of sickle cell disease

Blood ◽  
2004 ◽  
Vol 104 (1) ◽  
pp. 270-280 ◽  
Author(s):  
Maria L. Jison ◽  
Peter J. Munson ◽  
Jennifer J. Barb ◽  
Anthony F. Suffredini ◽  
Shefali Talwar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Steady State ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Expression Profiles ◽  
Ischemia Reperfusion ◽  
Transcriptional Analysis ◽  
Heme Oxygenase 1 ◽  
Specific Gene ◽  
Cell Disease

Abstract In sickle cell disease, deoxygenation of intra-erythrocytic hemoglobin S leads to hemoglobin polymerization, erythrocyte rigidity, hemolysis, and microvascular occlusion. Ischemia-reperfusion injury, plasma hemoglobin-mediated nitric oxide consumption, and free radical generation activate systemic inflammatory responses. To characterize the role of circulating leukocytes in sickle cell pathogenesis we performed global transcriptional analysis of blood mononuclear cells from 27 patients in steady-state sickle cell disease (10 patients treated and 17 patients untreated with hydroxyurea) compared with 13 control subjects. We used gender-specific gene expression to validate human microarray experiments. Patients with sickle cell disease demonstrated differential gene expression of 112 genes involved in heme metabolism, cell-cycle regulation, antioxidant and stress responses, inflammation, and angiogenesis. Inducible heme oxygenase-1 and downstream proteins biliverdin reductase and p21, a cyclin-dependent kinase, were up-regulated, potentially contributing to phenotypic heterogeneity and absence of atherosclerosis in patients with sickle cell disease despite endothelial dysfunction and vascular inflammation. Hydroxyurea therapy did not significantly affect leukocyte gene expression, suggesting that such therapy has limited direct anti-inflammatory activity beyond leukoreduction. Global transcriptional analysis of circulating leukocytes highlights the intense oxidant and inflammatory nature of steady-state sickle cell disease and provides insight into the broad compensatory responses to vascular injury.

scholarly journals Effects of Globin mRNA Reduction Methods on Gene Expression Profiles from Whole Blood

2006 ◽  
Vol 8 (5) ◽  
pp. 551-558 ◽  
Author(s):  
Jinny Liu ◽  
Elizabeth Walter ◽  
David Stenger ◽  
Dzung Thach
Keyword(s):  
Gene Expression ◽  
Whole Blood ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Globin Mrna ◽  
Reduction Methods

scholarly journals Whole Blood Gene Expression Profiles in Insulin Resistant Latinos with the Metabolic Syndrome

PLoS ONE ◽  
2013 ◽  
Vol 8 (12) ◽  
pp. e84002 ◽  
Author(s):  
Samantha E. Tangen ◽  
Darwin Tsinajinnie ◽  
Martha Nuñez ◽  
Gabriel Q. Shaibi ◽  
Lawrence J. Mandarino ◽  
...  
Keyword(s):  
Gene Expression ◽  
Metabolic Syndrome ◽  
Whole Blood ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Blood Gene Expression ◽  
Insulin Resistant ◽  
The Metabolic Syndrome

Genomic Approaches to Identifying Risk for Pulmonary Artery Hypertension among Individuals with Sickle Cell Disease.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1442-1442
Author(s):  
Damian Silbermins ◽  
Laura M. De Castro ◽  
Jude C Jonassaint ◽  
Shiaowen David Hsu ◽  
Marilyn J. Telen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sickle Cell Disease ◽  
Pulmonary Artery ◽  
Sickle Cell ◽  
Whole Blood ◽  
Gene Signature ◽  
Pulmonary Artery Hypertension ◽  
Gene Set Enrichment Analysis ◽  
Cell Disease ◽  
Gene Signatures

Abstract Pulmonary artery hypertension (PAH) occurs in 30–50% of adult patients with sickle cell disease (SCD), with mortality ranging from 16 to 50% and a median survival of 25 months. Our objective was to use gene expression profiling to develop a gene signature predictor for PAH through the analysis of gene expression of blood cells from SCD patients with or without PAH. We hypothesized that these gene signatures could allow us to identify patients at risk for PAH, as well as to generate hypotheses as to the pathophysiology of PAH in SCD. We used Affymetrix U133A2 GeneChip to determine the RNA expression of both whole blood and leukocytes using PAXgene and Leukolock methods, respectively. The study population included patients homozygous for HbS or with HbSβ0 thalassemia. Subjects with PAH were ≥18 years old, in steady state, and had PAH either by 2D echo (TR jet ≥ 2.7 m/sec) or right-sided catheterization (mean PA pressure ≥ 30 mmHg). Patients were excluded if they were pregnant, had co-existing rheumatologic conditions or other inflammatory diseases, were on chronic transfusion therapy or had had a vaso-occlusive episode in the previous 4 weeks. The control subjects were patients with SCD but without PAH (TR jet ≤ 1.8 m/sec or mean PA pressure &lt;25 mmHg). Hierarchical clustering based on the gene expression pattern from 7 patients with PAH and 6 controls showed a trend for the clustering of SCD patients with PAH away from SCD patients without PAH. This trend was present for the gene expression in both whole blood and leukocytes. A Bayesian regression analysis was then performed to identify a set of predictor gene signatures for the PAH phenotype (Figure 1) in SCD. Finally, using gene set enrichment analysis, we found that the leukocytes from patients with PAH were highly enriched in the gene sets deriving from hematopoietic stem cells, corroborating the hypothesis of hyperhemolysis and higher blood cell turnover in this population. Other pathways showing upregulation in PAH were PTEN, TGFβ, cyclin D1, WNT and PPAR. Although these data are preliminary, they suggest that PAH in SCD does indeed have a distinct gene signature profile that may become useful in identifying risk for PAH prospectively, as well as in directing further investigation into the pathogenesis of PAH in SCD. Figure Figure

scholarly journals Micro RNA Profiling to Identify Regulator of Fetal Hemoglobin Expression in Sickle Cell Disease

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 49-49 ◽  
Author(s):  
Biaoru Li ◽  
Christina M. Torres ◽  
Mayuko Takezaki ◽  
Cindy Neunert ◽  
Abdullah Kutlar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Fetal Hemoglobin ◽  
Mrna Levels ◽  
Cell Disease ◽  
Erythroid Progenitors ◽  
Globin Mrna ◽  
Mirna Genes

Abstract Elevated fetal hemoglobin (HbF) expression ameliorates the clinical severity of sickle cell disease (SCD) by inhibiting hemoglobin S polymerization. Differences in HbF levels are attributed to inherited DNA genetic variations that regulate γ-globin transcription; however the role of microRNA (miRNA) genes in HbF regulation has not been investigated using clinical samples. miRNAs are small non-protein-coding RNA molecules that negatively regulate gene expression through inhibition of mRNA translation. Our goal is to identify miRNA genes with altered expression in sickle cell patients with elevated HbF levels, to elucidate mechanisms of γ-globin gene regulation. After IRB approval, peripheral blood was collected from SCD patients (not on hydroxyurea therapy), followed in the pediatric and adult Sickle Cell Clinics at Georgia Regents University. HbF levels measured by high performance liquid chromatography and complete blood and reticulocyte counts were obtained. Twelve blood samples, six each from SCD subjects with high HbF (19.9±2.1%) or low HbF (4.4±0.9%) levels were analyzed. We observed more severe anemia and higher reticulocyte counts in the low HbF group. After Histopaque separation, red blood cells were processed on a MACS column with anti-CD71 antibody to isolate reticulocytes, followed by total RNA extraction using Trizol. RNA (750ng) was hybridized to a genome-wide miRCURY LNA microRNA Array (Exiqon) containing 1,921 human probes. The microarray raw data were collected on an Agilent G2565BA Microarray Scanner System and normalized by Model-Based Background Correction and Principle Component Analysis. Characterization of miRNA profiles for low HbF compared to high HbF groups identified 327 differentially expressed genes including multiple miR-144 isoforms. We subsequently explored the function of miR-144 because it targets Nrf2 which mediates drug-induced HbF expression and Nrf2 has an antioxidant protective effect in SCD. Therefore, we conducted supervised learning of the normalized microarray data based on miR-144 expression. Interestingly, in the low HbF group we observed two subphenotypes: 1) associated with 8-fold increased miR-144 expression (3 subjects) and 2) associated with no change in miR-144 level (3 subjects) when compared to the high HbF group suggesting a role of miR-144 in HbF regulation. In the supervised learning analysis, there were 62 up-regulated and 33 down-regulated miRNAs (&gt;2-fold; p&lt;0.05) in the first subphenotype. We hypothesized that miRNAs up-regulated in the low HbF group might silence known γ-globin trans-activators. By TargetScan and Miranda analysis 7 miRNAs were predicted to target γ-globin including miR-96 a known negative regulator. There were 4 miRNAs predicted to target Nrf2 and 12 miRNAs that target other transcription factors such as KLF1, KLF4, BCL11A, and GATA2. To define a functional role of miR-144 we conducted studies in adult CD34+ stem cells grown in a two-phase culture system containing Stem Cell Factor (50ng/mL), Interleukin-3 (10ng/mL) and Erythropoietin (4IU/mL). At day 8 in culture, miR-144 mimic, miR-144 antagomir (inhibitor) or scrambled control (100nM, 200nM, and 300nM) were transfected using a Nucleofector System. After 72 hr incubation, RT-qPCR was conducted to measure γ-globin and Nrf2 mRNA levels. miR-144 mimic or antagomir at 100-300nM concentrations had no significant effect on γ-globin mRNA levels. By contrast, flow cytometry analysis using a FITC-anti-γ-globin antibody in erythroid cells treated with miR-144 mimic, produced a 30-70% decrease in HbF positive cells (p&lt;0.05). On the contrary, we observed a 1.8-fold increase in HbF positive cells mediated by 300nM miR-144 antagomir. Evidence that miR-144 targets Nrf2 was established when antagomir treatment increased Nrf2 expression 1.4-fold (p&lt;0.05). Final studies using day 8 erythroid progenitors treated with Nrf2 siRNA demonstrated a 40% decrease in γ-globin mRNA supporting a role of Nrf2 on γ-gene expression. In summary, the miRNA profiles associated with HbF expression in SCD patients combined with functional studies in human primary erythroid progenitors, support a role for miR-144 in γ-globin regulation. These findings will be expanded to a pre-clinical SCD mouse model to develop miR-144 as a potential therapeutic option. Disclosures No relevant conflicts of interest to declare.

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