RNA Transcript Expression Research Articles

Abstract 311: The Long Non-Coding RNA H19 Regulates Experimental Abdominal Aortic Aneurysm Development and Progression

Background: Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators in various biological processes and diseases. Here we sought to identify and functionally characterize the lncRNA H19 as a novel regulator in abdominal aortic aneurysm development. Methods and Results: We profiled RNA transcript expression in two murine abdominal aortic aneurysm models, Angiotensin II (ANGII) infusion in ApoE-/- mice and porcine pancreatic elastase (PPE) instillation in C57BL/6 wildtype mice. The lncRNA H19 was identified as one of the most highly up-regulated transcripts in both mouse aneurysm models compared to sham-operated controls. This was confirmed by qRT-PCR and in situ hybridization. Experimental knock-down of H19, utilizing site-specific antisense oligonucleotides (LNA-GapmeRs) in vivo , significantly limited aneurysm growth in both models. Upregulated H19 correlated well with smooth muscle cell (SMC) content and its apoptosis in progressing aneurysms. Of importance, a similar pattern could be observed in human AAA tissue samples, and in a novel preclinical LDLR-/- Yucatan mini-pig aneurysm model. In vitro knock-down of H19 markedly decreased apoptotic rates of cultured human aortic SMCs, while overexpression of H19 had the opposite effect. Interestingly, H19-dependent cell fate decisions in SMCs appeared independent of miR-675, which is embedded in the first exon of the H19 gene. A customized transcription factor array and proteomic analysis revealed the hypoxia-inducible factor 1-alpha (HIF1A) and interleukin 1 receptor-like 2 (IL1RL2) as the main downstream effectors being regulated via H19. Conclusion: The lncRNA H19 is a novel regulator of SMC survival and aortic inflammation in AAA development and progression. Ultimately inhibition of H19 expression might serve as a novel molecular therapeutic target for aneurysm disease. Key words: Long noncoding RNAs (lncRNAs); abdominal aortic aneurysm; smooth muscle cells (SMCs); Inflammation

AKT2 is the Predominant Akt Isoform Expressed in Human Skeletal Muscle

AKT is a serine/threonine kinase that mediates a number of intracellular processes including proliferation, survival, and metabolism. Three Akt isoforms have been identified in mammals (AKT1, AKT2, and AKT3), and there exist distinct and redundant functions for each. While a great deal of our knowledge regarding the functions of the three AKT isoforms in skeletal muscle has been derived from the use of cell and animal models, there is limited information regarding the expression and actions of AKT isoforms in human skeletal muscle. Given this, we performed a series of experiments to determine the abundances of each AKT isoform in human skeletal muscle, and to identify the principal AKT isoform involved in human myogenesis. We first examined the RNA transcript expression of each AKT isoform from biopsies obtained from human quadriceps. AKT2 was the most highly expressed AKT transcript, exhibiting a15.5‐fold increase over AKT1 and AKT3 transcripts (P<0.001). No significant difference between expression of AKT1 and AKT3 was observed. We next determined the abundance of AKT protein isoforms by using antibody immunoprecipitation followed by Liquid Chromatography‐Parallel Reaction Monitoring/Mass Spectrometry (LC‐PRM/MS). Immunoprecipitation was performed using both mouse and rabbit pan AKT antibodies that were immunoreactive with purified peptides of all three AKT isoforms. Analysis of immunoprecipitates by LC‐PRM/MS revealed that AKT2 was the most highly expressed AKT in quadriceps muscle (4.2‐fold greater than AKT1 using the rabbit antibody (P<0.001) and 1.6‐fold greater than AKT1 using the mouse antibody(P<0.05)). Expression of AKT3 protein was virtually undetectable in quadriceps, suggesting that AKT3 plays little or no role in skeletal muscle physiology. Having found that AKT2 is the most highly expressed AKT isoform in human quadriceps, we next asked whether AKT2 was the principal isoform regulating human myotube formation. To test this, cultured primary human myoblasts were virally‐transduced with cDNAs encoding either wild‐type (WT) or kinase‐dead (KD) AKT1 or AKT2 and allowed to terminally differentiate. Myotubes expressing either WT‐AKT1 or WT‐AKT2 showed enhanced fusion compared to control myotubes(P<0.05 for each), while myotubes expressing KD‐AKT1 showed a 10% reduced fusion index (P<0.05). However, compared to control myotubes, myotubes expressing KD‐AKT2 showed a 63% decrease in fusion index (P<0.001). In conclusion, our findings identify AKT2 as the predominant AKT isoform expressed in human quadriceps muscle and as the principal AKT isoform that regulates differentiation of cultured human skeletal myoblasts.Support or Funding InformationThe opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Army or the Department of Defense.

RNA transcript expression of IGF-I/PI3K pathway components in regenerating skeletal muscle is sensitive to initial injury intensity

ObjectiveSkeletal muscle regeneration is a complex process involving the coordinated input from multiple stimuli. Of these processes, actions of the insulin-like growth factor-I (IGF-I) and phosphoinositide 3-kinase (PI3K) pathways are vital; however, whether IGF-I or PI3K expression is modified during regeneration relative to initial damage intensity is unknown. The objective of this study was to determine whether mRNA expression of IGF-I/PI3K pathway components was differentially regulated during muscle regeneration in mice in response to traumatic injury induced by freezing of two different durations. DesignTraumatic injury was imposed by applying a 6-mm diameter cylindrical steel probe, cooled to the temperature of dry ice (−79°C), to the belly of the left tibialis anterior muscle of 12-week-old C57BL/6J mice for either 5s (5s) or 10s (10s). The right leg served as the uninjured control. RNA was obtained from injured and control muscles following 3, 7, and 21days recovery and examined by real-time PCR. Expression of transcripts within the IGF, PI3K, and Akt families, as well as for myogenic regulatory factors and micro-RNAs were studied. ResultsThree days following injury, there was significantly increased expression of Igf1, Igf2, Igf1r, Igf2r, Pik3cb, Pik3cd, Pik3cg, Pik3r1, Pik3r5, Akt1, and Akt3 in response to either 5s or 10s injury compared to uninjured control muscle. There was a significantly greater expression of Pik3cb, Pik3cd, Pik3cg, Pik3r5, Akt1, and Akt3 in 10s injured muscle compared to 5s injured muscle. Seven days following injury, we observed significantly increased expression of Igf1, Igf2, Pik3cd, and Pik3cg in injured muscle compared to control muscle in response to 10s freeze injury. We also observed significantly reduced expression of Igf1r and miR-133a in response to 5s freeze injury compared to control muscle, and significantly reduced expression of Ckm, miR-1 and miR-133a in response to 10s freeze injury as compared to control. Twenty-one days following injury, 5s freeze-injured muscle exhibited significantly increased expression of Igf2, Igf2r, Pik3cg, Akt3, Myod1, Myog, Myf5, and miR-206 compared to control muscle, while 10s freeze-injured muscles showed significantly increased expression of Igf2, Igf2r, Pik3cb, Pik3cd, Pik3r5, Akt1, Akt3, and Myog compared to control. Expression of miR-1 was significantly reduced in 10s freeze-injured muscle compared to control muscle at this time. There were no significant differences in RNA expression between 5s and 10s injury at either 7d or 21d recovery in any transcript examined. ConclusionsDuring early skeletal muscle regeneration in mice, transcript expressions for some components of the IGF-I/PI3K pathway are sensitive to initial injury intensity induced by freeze damage.

Abstract 954: Analysis of noncoding RNA expression in a mouse model of PTEN-deficient prostate cancer

Abstract Prostate cancer is a heterogeneous disease that is driven by combined genetic and epigenetic alterations. A significant portion of the mammalian genome consists of non-coding regions of RNA. Increasing evidence has shown that these noncoding RNAs (ncRNAs) have significant roles in the epigenetic regulation of several cellular processes, and their dysregulation can contribute to tumorigenesis and promote disease progression in many cancer types. In order to gain better insights into the potential roles of ncRNAs in prostate cancer, we used a genetically engineered mouse model of prostate cancer to perform a comparative analysis of the cancer transcriptome and the landscape of ncRNAs in castration-naïve prostate tumors and the progression to castration-resistant disease. Whole transcription arrays were used to explore both messenger (mRNA) and long intergenic non-coding RNA (LincRNA) transcript expression in normal prostate tissue from wildtype mice and prostate tumors from conditional PTEN-knockout mice. Comparative analyses were performed between prostate tumor samples from castration-naïve mice and, at 4 weeks and 10 weeks post-surgical castration. Altered expression of ncRNAs was most commonly observed and accounted for 56.2% (2370/4216), 47.6% (4460/9366) and 41.57% (1545/3717) of the total genes differentially expressed between normal mouse prostate and prostate tumors from castration-naïve, 4 weeks post-castration and 10 weeks post-castration, respectively. We also profile the expression of relevant cancer-related lincRNAs present in these models. Overall, our analyses provide data to support a role of LincRNA dysregulation in the pathogenesis of PTEN-deficient prostate cancer, and suggest that these mouse models might provide a potential preclinical tool to test candidate prospective therapies targeting ncRNAs. Citation Format: Marco A. De Velasco, Yurie Kura, Kazuko Sakai, Yuji Hatanaka, Yoshihiko Fujita, Yosuke Togashi, Masato Terashima, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Analysis of noncoding RNA expression in a mouse model of PTEN-deficient prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 954.

Abstract 512: The Long Non-coding Rna MIAT Regulates Smooth Muscle Cell Proliferation and Macrophage Activity in Advanced Atherosclerotic Lesions

Background: Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators in various biological processes and diseases. Here we sought to identify and functionally characterize the lncRNA MIAT as a novel regulator in atherosclerotic plaque stability. Methods and results: We profiled RNA transcript expression in patients with advanced atherosclerotic lesions from the Biobank of Karolinska Endarterectomies (BiKE). By microarray analysis, lncRNA MIAT was identified as one of the most highly up-regulated non-coding RNAs in carotid plaques compared to iliac artery controls, which was confirmed by qRT-PCR and in situ hybridization. Additional in silico analysis indicated a substantial positive correlation of MIAT with markers of inflammation, apoptosis and matrix degradation in carotid plaques. Experimental knock-down of MIAT, utilizing site-specific antisense oligonucleotides (LNA-GapmeRs) not only markedly decreased proliferation and migration rates of cultured human carotid artery smooth muscle cells (hCASMCs), but also increased their levels of apoptosis. In addition, MIAT inhibition significantly impaired oxidized LDL (oxLDL) uptake of murine peritoneal as well as human monocyte-differentiated macrophages in vitro. In contrast, induction of MIAT expression by lipoprotein-a (LPa) treatment, displayed the opposite effect. Conditioned medium from macrophage cultures after MIAT knock-down substantially decreased hCASMC proliferation, indicating a potential involvement of MIAT in macrophage-SMC interactions during advanced stages of atherosclerosis. Conclusion: The lncRNA MIAT is a novel regulator of cellular processes in atherosclerosis and plaque stability, which influences SMC proliferation and apoptosis and interacts with disease-triggering macrophages.

Adaptive capability as indicated by behavioral and physiological responses, plasma HSP70 level, and PBMC HSP70 mRNA expression in Osmanabadi goats subjected to combined (heat and nutritional) stressors.

A study was conducted to assess the impact of heat and nutritional stress simultaneously on the adaptive capability as indicated by behavioral and physiological responses, plasma heat shock protein 70 (HSP70) level, and peripheral blood mononuclear cells (PBMC) HSP70 gene expression in goats. Twenty-four adult Osmanabadi bucks (average body weight (BW) 16.0kg) were used in the present study. The bucks were divided into four groups viz., C (n=6; control), HS (n=6; heat stress), NS (n=6; nutritional stress), and CS (n=6; combined stress). The study was conducted for a period of 45days. C and HS bucks had ad libitum access to their feed while NS and CS bucks were under restricted feed (30% intake of C bucks) to induce nutritional stress. The HS and CS bucks were exposed to solar radiation for 6h a day between 10:00 a.m. and 4:00 p.m. to induce heat stress. The data was analyzed using repeated measures analysis of variance. The standing time differed significantly (P<0.01) between ad libitum fed groups (C and HS) and restricted feeding groups (NS and CS). The highest (P<0.01) lying time was recorded in the CS group while the lowest in the C and HS groups. The highest (P<0.01) drinking frequency was also recorded in the CS group. Water intake recorded was significantly (P<0.01) higher in both the HS and CS groups. The highest respiration rate (RR), pulse rate (PR), and rectal temperature (RT) during the afternoon were also recorded in the CS group. Further, skin temperature of the head, flank, and scrotum during the afternoon was also higher (P<0.01) in the CS group. In addition, both plasma HSP70 concentration and PBMC HSP70 messenger RNA (mRNA) transcript expression were also significantly (P<0.01) higher in the CS group. It can be concluded from this study that when two stressors occur simultaneously, they may have severe impact on adaptive capabilities of Osmanabadi bucks as compared to that would occur individually. Further, the study indicated that lying time, drinking frequency, RR, RT, plasma HSP70, and PBMC HSP70 gene expression may act as ideal biological markers for assessing the impact of CS on adaptive capabilities in bucks.

Correlating quantitative real-time PCR to rapid diagnostic test and RNA transcript expression in isolated gametocytemia and asexual parasitemia of Plasmodium falciparum malaria.

BackgroundAt present, only microscopic examination of stained thick and thin blood smears for malaria can differentiate clinically relevant asexual parasitemia from clinically irrelevant isolated gametocytemia. Microscopy is time consuming, labour intensive, and requires significant technical expertise to perform. Simple and rapid tests that can distinguish asexual from isolated sexual parasitemia are needed.MethodsTo determine if parasitemia and cycle threshold (CT) values on Plasmodium genus and P. falciparum-specific quantitative polymerase chain reaction (qPCR) assays correlate to positivity of rapid diagnostic test (RDT), and 18S rRNA gene copy number, we analyzed blood samples from Ontario patients with isolated P. falciparum gametocytemia or asexual stages. RNA transcripts were evaluated to determine whether there is correlation of expression to different life cycle stages of P. falciparum.Results45 specimens containing isolated P. falciparum gametocytes, and 40 specimens containing isolated asexual stages by microscopy were identified and analyzed. By RDT, 40 of 45 (88.9 %) isolated gametocytemia specimens and 40 of 40 (100 %) asexual-stage specimens were positive for Plasmodium falciparum-specific histidine rich protein-2 (HRP-2). Fourteen of 45 (31.1 %) isolated gametocytemia specimens, and 36 of 40 (90 %) asexual-stage specimens were positive for Plasmodium genus aldolase T2 band. Positivity of the aldolase T2 band was associated with lower mean Plasmodium genus and P. falciparum-specific CT values, and to higher mean 18S rRNA gene copy by qPCR for both isolated gametocytemia and asexual-stage specimens. There was also a negative correlation of asexual parasitemia to both CT values, and positive correlation to 18S rRNA gene copy number. Analysis of asexual stage-specific erythrocyte binding antigen (eba-175) transcripts on 25 isolated gametocytemia and 20 asexual-stage specimens gave a positive predictive value of 62.5 % and negative predictive value of 100 % for asexual parasitemia. Thus, an absence of eba-175 transcripts excluded the presence of asexual (clinically relevant) parasitemia.ConclusionsPositivity of the aldolase T2 band of BinaxNow RDT correlated to higher parasite load in both isolated gametocytemia and asexual-stage specimens. Asexual stage-specific eba-175 RNA transcript expression provided reasonable negative predictive value for exclusion of asexual parasitemia in clinical samples, but was present in both isolated gametocytemia and asexual stage specimens.

Overlap of expression quantitative trait loci (eQTL) in human brain and blood.

BackgroundExpression quantitative trait loci (eQTL) are genomic regions regulating RNA transcript expression levels. Genome-wide Association Studies (GWAS) have identified many variants, often in non-coding regions, with unknown functions and eQTL provide a possible mechanism by which these variants may influence observable phenotypes. Limited access and availability of tissues such as brain has led to the use of blood as a substitute for eQTL analyses.MethodsHere, we evaluate the overlap of eQTL reported in published studies conducted in blood and brain tissues to assess the utility of blood as an alternative to brain tissue in the study of neurological and psychiatric conditions. Expression QTL results from eight published brain studies were compared to blood eQTL identified in from a meta-analysis involving 5,311 individuals. We accounted for differences in SNP platforms and study design by using SNP proxies in high linkage disequilibrium with reported eQTL. The degree of overlap between studies was calculated by ascertaining if an eQTL identified in one study was also identified in the other study.ResultsThe percentage of eQTL overlapping for brain and blood expression after adjusting for differences in sample size ranged from 13 - 23% (mean 19.2%). Amongst pairs of brain studies eQTL overlap ranged from 0 - 35%, with higher degrees of overlap found for studies using expression data collected from the same brain region.ConclusionOur results suggest that whenever possible tissue specific to the pathophysiology of the disease being studied should be used for transcription analysis.

Effects of Graded Taurine Levels on Juvenile Cobia

AbstractTaurine, which has multiple important physiological roles in teleost fish and mammals, is an amino acid not found in alternative protein sources not derived from animals. Although taurine is found in fish‐meal‐based feeds, its high water solubility leads to lower taurine levels in reduction‐process‐based feeds, which marine carnivores such as Cobia Rachycentron canadum are adapted to in their natural diets. Graded taurine supplementation (0, 0.5, 1.5, and 5.0%) added to a traditional fish‐meal‐based formulation was examined in two growth trials with Cobia: one initiated with 10‐g individuals and the second initiated with 120‐g individuals. During the first trial, in which growth as weight gain ranged from 123 to 139 g per fish, there was an increase in dietary taurine and a decrease in the feed conversion ratio from 1.04 to 0.99. During the second trial, in which growth ranged from 227 to 313 g gained per fish, there was no significant difference in performance characteristics between dietary treatments. Messenger RNA transcript expression levels for two of the genes involved in taurine synthesis, cysteine dioxygenase (CDO) and cysteamine dioxygenase (ADO), as well as the membrane‐bound taurine transporter, TauT, were also measured at the conclusion of the second trial. Increasing dietary taurine in a diet containing 34.5% fish meal did not result in significantly different growth or production characteristics in Cobia, but did result in significantly increased taurine levels in fillet, liver, and plasma.

Autoimmune susceptibility gene critically influences CD39 T cell expression and function in modulating human inflammation (P3313)

Abstract Background: Purinergic signaling is an important mechanism of immunomodulation. The ectoenzyme CD39 is a key regulator of extracellular ATP levels and catalyses hydrolysis of ATP to AMP. The A allele of the CD39 SNP rs10748643 is associated with both susceptibility to the autoimmune condition Crohn’s disease and low CD39 RNA transcript expression levels in HapMap transformed immune cell lines. Aim: To investigate the association between CD39 genetic polymorphism, protein expression and function in human healthy and autoimmune arthritis inflamed tissues. Results: We demonstrate increased CD39 protein on T cells in the human inflamed arthritis joint compared to peripheral blood. SNP rs10748643 AA individuals had a greatly reduced CD39 T cell expression and ATPase capacity compared to GG individuals (p&amp;lt;0.001). An electrophoretic mobility shift assay revealed a decrease in nuclear protein-DNA complex with rs10748643 AA probe compared to the GG probe. Bioinformatic analysis revealed AA individuals had ablation of an Sp1 transcription factor binding site, previously shown to be important in CD39 upregulation. Discussion: This is the first study to show how a specific SNP genotype, previously found in increased frequencies in patients with Crohn’s autoimmune disease and associated with reduced CD39 mRNA expression, is associated with functional modulation of the immune system.

Transcriptome analysis at four developmental stages of grape berry (Vitis vinifera cv. Shiraz) provides insights into regulated and coordinated gene expression

BackgroundVitis vinifera berry development is characterised by an initial phase where the fruit is small, hard and acidic, followed by a lag phase known as veraison. In the final phase, berries become larger, softer and sweeter and accumulate an array of organoleptic compounds. Since the physiological and biochemical makeup of grape berries at harvest has a profound impact on the characteristics of wine, there is great interest in characterising the molecular and biophysical changes that occur from flowering through veraison and ripening, including the coordination and temporal regulation of metabolic gene pathways. Advances in deep-sequencing technologies, combined with the availability of increasingly accurate V. vinifera genomic and transcriptomic data, have enabled us to carry out RNA-transcript expression analysis on a global scale at key points during berry development.ResultsA total of 162 million 100-base pair reads were generated from pooled Vitis vinifera (cv. Shiraz) berries sampled at 3-weeks post-anthesis, 10- and 11-weeks post-anthesis (corresponding to early and late veraison) and at 17-weeks post-anthesis (harvest). Mapping reads from each developmental stage (36-45 million) onto the NCBI RefSeq transcriptome of 23,720 V. vinifera mRNAs revealed that at least 75% of these transcripts were detected in each sample. RNA-Seq analysis uncovered 4,185 transcripts that were significantly upregulated at a single developmental stage, including 161 transcription factors. Clustering transcripts according to distinct patterns of transcription revealed coordination in metabolic pathways such as organic acid, stilbene and terpenoid metabolism. From the phenylpropanoid/stilbene biosynthetic pathway at least 46 transcripts were upregulated in ripe berries when compared to veraison and immature berries, and 12 terpene synthases were predominantly detected only in a single sample. Quantitative real-time PCR was used to validate the expression pattern of 12 differentially expressed genes from primary and secondary metabolic pathways.ConclusionsIn this study we report the global transcriptional profile of Shiraz grapes at key stages of development. We have undertaken a comprehensive analysis of gene families contributing to commercially important berry characteristics and present examples of co-regulation and differential gene expression. The data reported here will provide an invaluable resource for the on-going molecular investigation of wine grapes.

In Vitro and Ex Vivo Delivery of Short Hairpin RNAs for Control of Hepatitis C Viral Transcript Expression

Recurrent hepatitis C virus (HCV) infection is the most common cause of graft loss and patient death after transplantation for HCV cirrhosis. Transplant surgeons have access to uninfected explanted livers before transplantation and an opportunity to deliver RNA interference-based protective gene therapy to uninfected grafts. Conserved HCV sequences were used to design short interfering RNAs and test their ability to knockdown HCV transcript expression in an in vitro model, both by transfection and when delivered via an adeno-associated viral vector. In a rodent model of liver transplantation, portal venous perfusion of explanted grafts with an adeno-associated viral vector before transplantation produced detectable short hairpin RNA transcript expression after transplantation. The ability to deliver anti-HCV short hairpin RNAs to uninfected livers before transplantation and subsequent exposure to HCV offers hope for the possibility of preventing the currently inevitable subsequent infection of liver grafts with HCV.

FDM: a graph-based statistical method to detect differential transcription using RNA-seq data

Motivation: In eukaryotic cells, alternative splicing expands the diversity of RNA transcripts and plays an important role in tissue-specific differentiation, and can be misregulated in disease. To understand these processes, there is a great need for methods to detect differential transcription between samples. Our focus is on samples observed using short-read RNA sequencing (RNA-seq).Methods: We characterize differential transcription between two samples as the difference in the relative abundance of the transcript isoforms present in the samples. The magnitude of differential transcription of a gene between two samples can be measured by the square root of the Jensen Shannon Divergence (JSD*) between the gene's transcript abundance vectors in each sample. We define a weighted splice-graph representation of RNA-seq data, summarizing in compact form the alignment of RNA-seq reads to a reference genome. The flow difference metric (FDM) identifies regions of differential RNA transcript expression between pairs of splice graphs, without need for an underlying gene model or catalog of transcripts. We present a novel non-parametric statistical test between splice graphs to assess the significance of differential transcription, and extend it to group-wise comparison incorporating sample replicates.Results: Using simulated RNA-seq data consisting of four technical replicates of two samples with varying transcription between genes, we show that (i) the FDM is highly correlated with JSD* (r=0.82) when average RNA-seq coverage of the transcripts is sufficiently deep; and (ii) the FDM is able to identify 90% of genes with differential transcription when JSD* >0.28 and coverage >7. This represents higher sensitivity than Cufflinks (without annotations) and rDiff (MMD), which respectively identified 69 and 49% of the genes in this region as differential transcribed. Using annotations identifying the transcripts, Cufflinks was able to identify 86% of the genes in this region as differentially transcribed. Using experimental data consisting of four replicates each for two cancer cell lines (MCF7 and SUM102), FDM identified 1425 genes as significantly different in transcription. Subsequent study of the samples using quantitative real time polymerase chain reaction (qRT-PCR) of several differential transcription sites identified by FDM, confirmed significant differences at these sites.Availability: http://csbio-linux001.cs.unc.edu/nextgen/software/FDMContact: darshan@email.unc.eduSupplementary information: Supplementary data are available at Bioinformatics online.

Recombinant erythroid Kruppel-like factor fused to GATA1 up-regulates delta- and gamma-globin expression in erythroid cells

AbstractThe β-hemoglobinopathies sickle cell disease and β-thalassemia are among the most common human genetic disorders worldwide. Hemoglobin A2 (HbA2, α2δ2) and fetal hemoglobin (HbF, α2γ2) both inhibit the polymerization of hemoglobin S, which results in erythrocyte sickling. Expression of erythroid Kruppel-like factor (EKLF) and GATA1 is critical for transitioning hemoglobin from HbF to hemoglobin A (HbA, α2β2) and HbA2. The lower levels of δ-globin expression compared with β-globin expression seen in adulthood are likely due to the absence of an EKLF-binding motif in the δ-globin proximal promoter. In an effort to up-regulate δ-globin to increase HbA2 expression, we created a series of EKLF-GATA1 fusion constructs composed of the transactivation domain of EKLF and the DNA-binding domain of GATA1, and then tested their effects on hemoglobin expression. EKLF-GATA1 fusion proteins activated δ-, γ-, and β-globin promoters in K562 cells, and significantly up-regulated δ- and γ-globin RNA transcript and protein expression in K562 and/or CD34+ cells. The binding of EKLF-GATA1 fusion proteins at the GATA1 consensus site in the δ-globin promoter was confirmed by chromatin immunoprecipitation assay. Our studies demonstrate that EKLF-GATA1 fusion proteins can enhance δ-globin expression through interaction with the δ-globin promoter, and may represent a new genetic therapeutic approach to β-hemoglobinopathies.

Immunohistochemical detection of ZAP70 in chronic lymphocytic leukemia predicts immunoglobulin heavy chain gene mutation status and time to progression

Zeta-associated protein-70 (ZAP70) expression measured by flow cytometry has been proposed as a surrogate marker of the somatic mutation status of the immunoglobulin heavy chain variable region (IGHV) genes in chronic lymphocytic leukemia. However, attempts to implement this approach in clinical flow cytometry laboratories have been problematic; many commercially available antibodies give unreliable results. Assessment of ZAP70 protein expression by immunohistochemistry in chronic lymphocytic leukemia tissue sections is an easy, alternative approach, although lack of quantitation and subjective interpretation of results are potential pitfalls. In this study, we correlated ZAP70 protein expression, assessed by immunohistochemistry, with ZAP70 messenger RNA (mRNA) transcript expression, assessed by semi-quantitative real-time reverse transcriptase–polymerase chain reaction assay, with the somatic mutation status of the IGHV genes in previously untreated patients with chronic lymphocytic leukemia. Expression of ZAP70 protein and mRNA transcripts correlated strongly (P=8.238 × 10−12). Expression of ZAP70 protein and mRNA transcripts also correlated strongly with the somatic mutation status of the IGHV genes (P=0.000071 and P=0.00076, respectively). Further, ZAP70 positivity by immunohistochemistry was associated with an increased risk of progression to therapy requirement (3-year risk 83% vs 31% for ZAP70 negative by immunohistochemistry, P=0.03). These results show that ZAP70 expression assessed by immunohistochemistry is a reliable surrogate marker of the somatic mutation status of the IGHV genes, and predicts time to progression.

Cis‐acting Allelic Variation in MUC5B mRNA Expression is Associated with Different Promoter Haplotypes

Mucins play a critical role in protecting and clearing the airways of noxious materials. Genetic variation that influences this environmental response is likely to affect respiratory disease susceptibility. It has been reported previously that variation in the promoter of MUC5B, the gene that encodes one of the two major gel-forming mucins (MUC5B) of the mucus of the respiratory tract, is associated with susceptibility to diffuse panbronchiolitis, and that a genetic difference in promoter activity can be detected in vitro. The aim of this study was to determine whether this genetic difference in promoter activity can be detected in vivo. Here, we undertake RNA transcript expression studies, making use of human fetal tissue to explore constitutive differences. We compare in vivo transcript expression levels in heterozygotes and use the Bayesian method, PHASE to associate exonic simple nucleotide polymorphisms (SNPs) with promoter SNPs to generate haplotypes. We successfully show haplotypic differences in MUC5B expression in vivo. This genetic variation should be taken into account in future studies on MUC5B in respiratory disease.

Treatment of Recalcitrant Generalized Morphea With Infliximab

A 66-year-old white woman with a history of hypertension and degenerative arthritis was seen in July 2003 by her primary care physician (PCP) for erythematous and sclerotic patches on her trunk. A skin biopsy specimen taken by the PCP at that time was interpreted (by an outside pathology laboratory) to indicate granuloma annulare. The patient began treatment with systemic corticosteroids and ceased taking her antihypertensive medication. However, her cutaneous symptoms continued to progress, and she was referred to our clinic in January 2004. At that time, the patient denied any illnesses prior to her skin eruption and noted that the only change in her medications was from lisinopril to valsartan. A review of systems found no shortness of breath, dysphagia, worsening of chronic joint symptoms, or Raynaud phenomenon. The patient was noted to have extensive white sclerotic patches and a few eczematous inflammatory patches on the upper chest, breasts (excluding the areola), upper abdomen, flanks, and proximal extremities (Figure 1). Her skin was firm and sclerotic on palpation. No sclerodactyly was present. A skin biopsy specimen was obtained. Light microscopic examination revealed a striking pandermal sclerosing reaction. The collagen bundles were of wider caliber and oriented parallel to the long axis of the epidermis. In addition, the overlying epidermis was attenuated with prominent hyperkeratosis. The subjacent papillary dermis had a hyalinized appearance with vascular dropout. The findings were compatible with an overlap between morphea and lichen sclerosus et atrophicus (LS&A). In situ hybridization studies demonstrated focal staining of the endothelium for cytomegalovirus (CMV) and tumor necrosis factor (TNF) RNA transcript expression. Direct immunofluorescence showed deposits of C5b-9 within the microvasculature most compatible with a humorally mediated microangiopathy syndrome (Figure 2). Serologic studies revealed positive anti-CMV IgM and IgG. However, the findings of antinuclear antibody assays, extractable nuclear protein antibody tests, an interstitial lung battery, complete blood cell count, and comprehensive chemical analysis were all within normal limits, as was the erythrocyte sedimentation rate. The patient was also found to be very hypertensive and resumed taking antihypertensive medications along with class 1 topical corticosteroids. Figure 1. Eczematous inflammatory patches.

Statistical Tests for Detecting Differential RNA-Transcript Expression from Read Counts

AbstractAs a fruit of the current revolution in sequencing technology, transcriptomes can now be analyzed at an unprecedented level of detail. These advances have been exploited for detecting differential expressed genes across biological samples and for quantifying the abundances of various RNA transcripts within one gene. However, explicit strategies for detecting the hidden differential abundances of RNA transcripts in biological samples have not been defined. In this work, we present two novel statistical tests to address this issue: a 'gene structure sensitive' Poisson test for detecting differential expression when the transcript structure of the gene is known, and a kernel-based test called Maximum Mean Discrepancy when it is unknown. We analyzed the proposed approaches on simulated read data for two artificial samples as well as on factual reads generated by the Illumina Genome Analyzer for two C. elegans samples. Our analysis shows that the Poisson test identifies genes with differential transcript expression considerably better that previously proposed RNA transcript quantification approaches for this task. The MMD test is able to detect a large fraction (75%) of such differential cases without the knowledge of the annotated transcripts. It is therefore well-suited to analyze RNA-Seq experiments when the genome annotations are incomplete or not available, where other approaches have to fail.

Statistical Tests for Detecting Differential RNA-Transcript Expression from Read Counts

As a fruit of the current revolution in sequencing technology, transcriptomes can now be analyzed at an unprecedented level of detail. These advances have been exploited for detecting differential expressed genes across biological samples and for quantifying the abundances of various RNA transcripts within one gene. However, explicit strategies for detecting the hidden differential abundances of RNA transcripts in biological samples have not been defined. In this work, we present two novel statistical tests to address this issue: a 'gene structure sensitive' Poisson test for detecting differential expression when the transcript structure of the gene is known, and a kernel-based test called Maximum Mean Discrepancy when it is unknown. We analyzed the proposed approaches on simulated read data for two artificial samples as well as on factual reads generated by the Illumina Genome Analyzer for two C. elegans samples. Our analysis shows that the Poisson test identifies genes with differential transcript expression considerably better that previously proposed RNA transcript quantification approaches for this task. The MMD test is able to detect a large fraction (75%) of such differential cases without the knowledge of the annotated transcripts. It is therefore well-suited to analyze RNA-Seq experiments when the genome annotations are incomplete or not available, where other approaches have to fail.

Changes in retinal αB-crystallin (cryab) RNA transcript levels during periods of altered ocular growth in chickens

Changes in retinal crystallin gene expression have been implicated in the development of myopia in animal models. We therefore investigated the expression of αB-crystallin (cryab) in the chicken retina during periods of increased ocular growth induced by form-deprivation and negative lens-wear, and during periods of decreased ocular growth induced by diffuser removal from previously form-deprived eyes, and plus lens-wear. Cryab RNA transcript levels in the chicken retina were measured using semi-quantitative real-time RT-PCR, at times between 1 h and 10 days after the fitting of diffusers or negative lenses, and at times between 1 h and 3 days following the removal of diffusers from previously form-deprived eyes, or the addition of plus lenses. Changes in expression for each condition at each time-point are analysed relative to expression in retinas from age-matched untreated control birds. No change in relative expression of cryab RNA transcript was detected 1 h after fitting diffusers to induce form-deprivation myopia. A transient increase in cryab RNA transcript expression was detected around 1 day later (p = 0.02), but expression returned to control levels after three days. After 7 (p = 0.005) and 10 (p = 0.001) days, retinal cryab RNA transcript expression progressively increased relative to controls. After removal of the diffusers, to initiate recovery, cryab RNA transcript expression remained elevated, with only a slight return to control levels. During the development of lens-induced myopia, no changes in cryab RNA transcript expression relative to controls were seen on day 1, but increases were seen at 10 days (p = 0.004). No significant changes in retinal cryab RNA transcript expression were seen in response to plus lenses compared to either contralateral control values (MANOVA; F = 0.60, p = 0.48) or age-matched untreated values (MANOVA; F = 4.10, p = 0.08). Changes in retinal cryab RNA transcript expression were not systematically related to changes in the rate of eye growth. The role of the transient increase in cryab expression observed after 1 day of form-deprivation, which was not seen after fitting negative lenses, is unclear. The later increases in relative cryab expression seen during the development of form-deprivation and lens-induced myopia occur too late to have a major role in the differential regulation of eye growth between experimental and control eyes. Given that cryab is a member of the small heat shock protein family, the later increases may reflect the emergence of cell damage related to high myopic pathology in the experimentally enlarged eyes and retina.