Yanqin Yang

A systematic comparison and evaluation of high density exon arrays and RNA-seq technology used to unravel the peripheral blood transcriptome of sickle cell disease

BackgroundTranscriptomic studies in clinical research are essential tools for deciphering the functional elements of the genome and unraveling underlying disease mechanisms. Various technologies have been developed to deduce and quantify the transcriptome including hybridization and sequencing-based approaches. Recently, high density exon microarrays have been successfully employed for detecting differentially expressed genes and alternative splicing events for biomarker discovery and disease diagnostics. The field of transcriptomics is currently being revolutionized by high throughput DNA sequencing methodologies to map, characterize, and quantify the transcriptome.MethodsIn an effort to understand the merits and limitations of each of these tools, we undertook a study of the transcriptome in sickle cell disease, a monogenic disease comparing the Affymetrix Human Exon 1.0 ST microarray (Exon array) and Illumina’s deep sequencing technology (RNA-seq) on whole blood clinical specimens.ResultsAnalysis indicated a strong concordance (R = 0.64) between Exon array and RNA-seq data at both gene level and exon level transcript expression. The magnitude of differential expression was found to be generally higher in RNA-seq than in the Exon microarrays. We also demonstrate for the first time the ability of RNA-seq technology to discover novel transcript variants and differential expression in previously unannotated genomic regions in sickle cell disease. In addition to detecting expression level changes, RNA-seq technology was also able to identify sequence variation in the expressed transcripts.ConclusionsOur findings suggest that microarrays remain useful and accurate for transcriptomic analysis of clinical samples with low input requirements, while RNA-seq technology complements and extends microarray measurements for novel discoveries.

Telomere attrition and candidate gene mutations preceding monosomy 7 in aplastic anemia

The pathophysiology of severe aplastic anemia (SAA) is immune-mediated destruction of hematopoietic stem and progenitor cells (HSPCs). Most patients respond to immunosuppressive therapies, but a minority transform to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), frequently associated with monosomy 7 (-7). Thirteen SAA patients were analyzed for acquired mutations in myeloid cells at the time of evolution to -7, and all had a dominant HSPC clone bearing specific acquired mutations. However, mutations in genes associated with MDS/AML were present in only 4 cases. Patients who evolved to MDS and AML showed marked progressive telomere attrition before the emergence of -7. Single telomere length analysis confirmed accumulation of short telomere fragments of individual chromosomes. Our results indicate that accelerated telomere attrition in the setting of a decreased HSPC pool is characteristic of early myeloid oncogenesis, specifically chromosome 7 loss, in MDS/AML after SAA, and provides a possible mechanism for development of aneuploidy.

Iron, inflammation, and early death in adults with sickle cell disease

Rationale: Patients with sickle cell disease (SCD) have markers of chronic inflammation, but the mechanism of inflammation and its relevance to patient survival are unknown. Objective: To assess the relationship between iron, inflammation, and early death in SCD. Methods and Results: Using peripheral blood mononuclear cell transcriptome profile hierarchical clustering, we classified 24 patients and 10 controls in clusters with significantly different expression of genes known to be regulated by iron. Subsequent gene set enrichment analysis showed that many genes associated with the high iron cluster were involved in the toll-like receptor system (TLR4, TLR7, and TLR8) and inflammasome complex pathway (NLRP3, NLRC4, and CASP1). Quantitative PCR confirmed this classification and showed that ferritin light chain, TLR4, and interleukin-6 expression were >100-fold higher in patients than in controls (P<0.001). Further linking intracellular iron and inflammation, 14 SCD patients with a ferroportin Q248H variant that causes intracellular iron accumulation had significantly higher levels of interleukin-6 and C-reactive protein compared with 14 matched SCD patients with the wild-type allele (P<0.05). Finally, in a cohort of 412 patients followed for a median period of 47 months (interquartile range, 24–82), C-reactive protein was strongly and independently associated with early death (hazard ratio, 3.0; 95% confidence interval, 1.7–5.2; P<0.001). Conclusions: Gene expression markers of high intracellular iron in patients with SCD are associated with markers of inflammation and mortality. The results support a model in which intracellular iron promotes inflammatory pathways, such as the TLR system and the inflammasome, identifying important new pathways for additional investigation.

Heme-bound iron activates placenta growth factor in erythroid cells via erythroid Krüppel-like factor

In adults with sickle cell disease (SCD), markers of iron burden are associated with excessive production of the angiogenic protein placenta growth factor (PlGF) and high estimated pulmonary artery pressure. Enforced PlGF expression in mice stimulates production of the potent vasoconstrictor endothelin-1, producing pulmonary hypertension. We now demonstrate heme-bound iron (hemin) induces PlGF mRNA >200-fold in a dose- and time-dependent fashion. In murine and human erythroid cells, expression of erythroid Krüppel-like factor (EKLF) precedes PlGF, and its enforced expression in human erythroid progenitor cells induces PlGF mRNA. Hemin-induced expression of PlGF is abolished in EKLF-deficient murine erythroid cells but rescued by conditional expression of EKLF. Chromatin immunoprecipitation reveals that EKLF binds to the PlGF promoter region. SCD patients show higher level expression of both EKLF and PlGF mRNA in circulating blood cells, and markers of iron overload are associated with high PlGF and early mortality. Finally, PlGF association with iron burden generalizes to other human diseases of iron overload. Our results demonstrate a specific mechanistic pathway induced by excess iron that is linked in humans with SCD and in mice to markers of vasculopathy and pulmonary hypertension. These trials were registered at www.clinicaltrials.gov as #NCT00007150, #NCT00023296, #NCT00081523, and #NCT00352430.

Global intron retention mediated gene regulation during CD4+ T cell activation

T cell activation is a well-established model for studying cellular responses to exogenous stimulation. Using strand-specific RNA-seq, we observed that intron retention is prevalent in polyadenylated transcripts in resting CD4+ T cells and is significantly reduced upon T cell activation. Several lines of evidence suggest that intron-retained transcripts are less stable than fully spliced transcripts. Strikingly, the decrease in intron retention (IR) levels correlate with the increase in steady-state mRNA levels. Further, the majority of the genes upregulated in activated T cells are accompanied by a significant reduction in IR. Of these 1583 genes, 185 genes are predominantly regulated at the IR level, and highly enriched in the proteasome pathway, which is essential for proper T cell proliferation and cytokine release. These observations were corroborated in both human and mouse CD4+ T cells. Our study revealed a novel post-transcriptional regulatory mechanism that may potentially contribute to coordinated and/or quick cellular responses to extracellular stimuli such as an acute infection.

Characterization of the platelet transcriptome by RNA sequencing in patients with acute myocardial infarction

Abstract Transcripts in platelets are largely produced in precursor megakaryocytes but remain physiologically active as platelets translate RNAs and regulate protein/RNA levels. Recent studies using transcriptome sequencing (RNA-seq) characterized the platelet transcriptome in limited number of non-diseased individuals. Here, we expand upon these RNA-seq studies by completing RNA-seq in platelets from 32 patients with acute myocardial infarction (MI). Our goals were to characterize the platelet transcriptome using a population of patients with acute MI and relate gene expression to platelet aggregation measures and ST-segment elevation MI (STEMI) (n = 16) vs. non-STEMI (NSTEMI) (n = 16) subtypes. Similar to other studies, we detected 9565 expressed transcripts, including several known platelet-enriched markers (e.g. PPBP, OST4). Our RNA-seq data strongly correlated with independently ascertained platelet expression data and showed enrichment for platelet-related pathways (e.g. wound response, hemostasis, and platelet activation), as well as actin-related and post-transcriptional processes. Several transcripts displayed suggestively higher (FBXL4, ECHDC3, KCNE1, TAOK2, AURKB, ERG, and FKBP5) and lower (MIAT, PVRL3, and PZP) expression in STEMI platelets compared to NSTEMI. We also identified transcripts correlated with platelet aggregation to TRAP (ATP6V1G2, SLC2A3), collagen (CEACAM1, ITGA2), and ADP (PDGFB, PDGFC, ST3GAL6). Our study adds to current platelet gene expression resources by providing transcriptome-wide analyses in platelets isolated from patients with acute MI. In concert with prior studies, we identify various genes for further study in regards to platelet function and acute MI. Future platelet RNA-seq studies examining more diverse sets of healthy and diseased samples will add to our understanding of platelet thrombotic and non-thrombotic functions.

Integrated analysis of miRNA and mRNA during differentiation of human CD34+ cells delineates the regulatory roles of microRNA in hematopoiesis

In the process of human hematopoiesis, precise regulation of the expression of lineage-specific gene products is critical for multiple cell-fate decisions that govern cell differentiation, proliferation, and self-renewal. Given the important role of microRNAs (miRNAs) in development and differentiation, we examined the global expression of miRNA in CD34(+) cells during lineage specific hematopoiesis and found 49 miRNAs to be differentially expressed, with functional roles in cellular growth and proliferation, and apoptosis. miR-18a was upregulated during erythropoiesis and downregulated during megakaryopoiesis. miR-145 was upregulated during granulopoiesis and down regulated during erythropoiesis. Megakaryopoitic differentiation resulted in significant alteration in the expression of many miRNAs that are believed to play critical roles in the regulation of B and T cell differentiation. Target prediction analyses on three different miRNA databases indicated that TargetScan outperformed microCosm and miRDB in identifying potential miRNA targets associated with hematopoietic differentiation process. An integrated analysis of the observed miRNAs and messenger RNAs (mRNAs) resulted in 87 highly correlated miRNA-mRNA pairs that have major functional roles in cellular growth and proliferation, hematopoietic system development, and Wnt/B-catenin and Flt 3 signaling pathways. We believe that this study will enhance our understanding on the regulatory roles of miRNA in hematopoiesis by providing a library of mRNA-miRNA networks.

A Role for Phosphodiesterase 3B in Acquisition of Brown Fat Characteristics by White Adipose Tissue in Male Mice

Obesity is linked to various diseases, including insulin resistance, diabetes, and cardiovascular disorders. The idea of inducing white adipose tissue (WAT) to assume characteristics of brown adipose tissue (BAT), and thus gearing it to fat burning instead of storage, is receiving serious consideration as potential treatment for obesity and related disorders. Phosphodiesterase 3B (PDE3B) links insulin- and cAMP-signaling networks in tissues associated with energy metabolism, including WAT. We used C57BL/6 PDE3B knockout (KO) mice to elucidate mechanisms involved in the formation of BAT in epididymal WAT (EWAT) depots. Examination of gene expression profiles in PDE3B KO EWAT revealed increased expression of several genes that block white and promote brown adipogenesis, such as C-terminal binding protein, bone morphogenetic protein 7, and PR domain containing 16, but a clear BAT-like phenotype was not completely induced. However, acute treatment of PDE3B KO mice with the β3-adrenergic agonist, CL316243, markedly increased the expression of cyclooxygenase-2, which catalyzes prostaglandin synthesis and is thought to be important in the formation of BAT in WAT and the elongation of very long-chain fatty acids 3, which is linked to BAT recruitment upon cold exposure, causing a clear shift toward fat burning and the induction of BAT in KO EWAT. These data provide insight into the mechanisms of BAT formation in mouse EWAT, suggesting that, in a C57BL/6 background, an increase in cAMP, caused by ablation of PDE3B and administration of CL316243, may promote differentiation of prostaglandin-responsive progenitor cells in the EWAT stromal vascular fraction into functional brown adipocytes.

The Very Low Density Lipoprotein Receptor Attenuates House Dust Mite–Induced Airway Inflammation by Suppressing Dendritic Cell–Mediated Adaptive Immune Responses

The very low density lipoprotein receptor (VLDLR) is a member of the low-density lipoprotein receptor family that binds multiple ligands and plays a key role in brain development. Although the VLDLR mediates pleiotropic biological processes, only a limited amount of information is available regarding its role in adaptive immunity. In this study, we identify an important role for the VLDLR in attenuating house dust mite (HDM)-induced airway inflammation in experimental murine asthma. We show that HDM-challenged Vldlr−/− mice have augmented eosinophilic and lymphocytic airway inflammation with increases in Th2 cytokines, C-C chemokines, IgE production, and mucous cell metaplasia. A genome-wide analysis of the lung transcriptome identified that mRNA levels of CD209e (DC-SIGNR4), a murine homolog of DC-SIGN, were increased in the lungs of HDM-challenged Vldlr−/− mice, which suggested that the VLDLR might modify dendritic cell (DC) function. Consistent with this, VLDLR expression by human monocyte-derived DCs was increased by HDM stimulation. In addition, 55% of peripheral blood CD11c+ DCs from individuals with allergy expressed VLDLR under basal conditions. Lastly, the adoptive transfer of HDM-pulsed, CD11c+ bone marrow–derived DCs (BMDCs) from Vldlr−/− mice to the airways of wild type recipient mice induced augmented eosinophilic and lymphocytic airway inflammation upon HDM challenge with increases in Th2 cytokines, C-C chemokines, IgE production, and mucous cell metaplasia, as compared with the adoptive transfer of HDM-pulsed, CD11c+ BMDCs from wild type mice. Collectively, these results identify a novel role for the VLDLR as a negative regulator of DC-mediated adaptive immune responses in HDM-induced allergic airway inflammation.

A Systems Biology Approach to Investigating Sex Differences in Cardiac Hypertrophy

Background Heart failure preceded by hypertrophy is a leading cause of death, and sex differences in hypertrophy are well known, although the basis for these sex differences is poorly understood. Methods and Results This study used a systems biology approach to investigate mechanisms underlying sex differences in cardiac hypertrophy. Male and female mice were treated for 2 and 3 weeks with angiotensin II to induce hypertrophy. Sex differences in cardiac hypertrophy were apparent after 3 weeks of treatment. RNA sequencing was performed on hearts, and sex differences in mRNA expression at baseline and following hypertrophy were observed, as well as within‐sex differences between baseline and hypertrophy. Sex differences in mRNA were substantial at baseline and reduced somewhat with hypertrophy, as the mRNA differences induced by hypertrophy tended to overwhelm the sex differences. We performed an integrative analysis to identify mRNA networks that were differentially regulated in the 2 sexes by hypertrophy and obtained a network centered on PPARα (peroxisome proliferator‐activated receptor α). Mouse experiments further showed that acute inhibition of PPARα blocked sex differences in the development of hypertrophy. Conclusions The data in this study suggest that PPARα is involved in the sex‐dimorphic regulation of cardiac hypertrophy.