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2.  Cytoplasmic sequestration of FUS/TLS associated with ALS alters histone marks through loss of nuclear protein arginine methyltransferase 1 
Human Molecular Genetics  2014;24(3):773-786.
Mutations in the RNA-binding protein FUS/TLS (FUS) have been linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Although predominantly nuclear, this heterogenous nuclear ribonuclear protein (hnRNP) has multiple functions in RNA processing including intracellular trafficking. In ALS, mutant or wild-type (WT) FUS can form neuronal cytoplasmic inclusions. Asymmetric arginine methylation of FUS by the class 1 arginine methyltransferase, protein arginine methyltransferase 1 (PRMT1), regulates nucleocytoplasmic shuttling of FUS. In motor neurons of primary spinal cord cultures, redistribution of endogenous mouse and that of ectopically expressed WT or mutant human FUS to the cytoplasm led to nuclear depletion of PRMT1, abrogating methylation of its nuclear substrates. Specifically, hypomethylation of arginine 3 of histone 4 resulted in decreased acetylation of lysine 9/14 of histone 3 and transcriptional repression. Distribution of neuronal PRMT1 coincident with FUS also was detected in vivo in the spinal cord of FUSR495X transgenic mice. However, nuclear PRMT1 was not stable postmortem obviating meaningful evaluation of ALS autopsy cases. This study provides evidence for loss of PRMT1 function as a consequence of cytoplasmic accumulation of FUS in the pathogenesis of ALS, including changes in the histone code regulating gene transcription.
PMCID: PMC4291251  PMID: 25274782
3.  New Noncoding Lytic Transcripts Derived from the Epstein-Barr Virus Latency Origin of Replication, oriP, Are Hyperedited, Bind the Paraspeckle Protein, NONO/p54nrb, and Support Viral Lytic Transcription 
Journal of Virology  2015;89(14):7120-7132.
We have previously shown that the Epstein-Barr virus (EBV) likely encodes hundreds of viral long noncoding RNAs (vlncRNAs) that are expressed during reactivation. Here we show that the EBV latency origin of replication (oriP) is transcribed bi-directionally during reactivation and that both leftward (oriPtLs) and rightward (oriPtRs) transcripts are largely localized in the nucleus. While the oriPtLs are most likely noncoding, at least some of the oriPtRs contain the BCRF1/vIL10 open reading frame. Nonetheless, oriPtR transcripts with long 5′ untranslated regions may partially serve noncoding functions. Both oriPtL and oriPtR transcripts are expressed with late kinetics, and their expression is inhibited by phosphonoacetic acid. RNA sequencing (RNA-seq) analysis showed that oriPtLs and oriPtRs exhibited extensive “hyperediting” at their Family of Repeat (FR) regions. RNA secondary structure prediction revealed that the FR region of both oriPtLs and oriPtRs may form large evolutionarily conserved and thermodynamically stable hairpins. The double-stranded RNA-binding protein and RNA-editing enzyme ADAR was found to bind to oriPtLs, likely facilitating editing of the FR hairpin. Further, the multifunctional paraspeckle protein, NONO, was found to bind to oriPt transcripts, suggesting that oriPts interact with the paraspeckle-based innate antiviral immune pathway. Knockdown and ectopic expression of oriPtLs showed that it contributes to global viral lytic gene expression and viral DNA replication. Together, these results show that these new vlncRNAs interact with cellular innate immune pathways and that they help facilitate progression of the viral lytic cascade.
IMPORTANCE Recent studies have revealed that the complexity of lytic herpesviral transcriptomes is significantly greater than previously appreciated with hundreds of viral long noncoding RNAs (vlncRNAs) being recently discovered. Work on cellular lncRNAs over the past several years has just begun to give us an initial appreciation for the array of functions they play in complex formation and regulatory processes in the cell. The newly identified herpesvirus lncRNAs are similarly likely to play a variety of different functions, although these functions are likely tailored to specific needs of the viral infection cycles. Here we describe novel transcripts derived from the EBV latency origin of replication. We show that they are hyperedited, that they interact with a relatively newly appreciated antiviral pathway, and that they play a role in facilitating viral lytic gene expression. These investigations are a starting point to unraveling the complex arena of vlncRNA function in herpesvirus lytic replication.
PMCID: PMC4473578  PMID: 25926645
Neuro-Oncology  2014;16(Suppl 5):v103.
Glioblastoma multiforme (GBM) is a devastating disease with poor survival rates. Despite years of research, there has been little change in the survival rate or median survival time. HCMV (human cytomegalovirus) was reported in GBM over a decade ago and this finding has the potential to increase our understanding of the disease and it offers a potential tumor specific therapeutic target. Because of this promise, there is a fair amount of time, energy and money being directed towards understanding and utilizing this connection for eventual therapeutic purposes. Despite these ongoing efforts and the time lapsed since the discovery of HCMV in GBM, the association remains controversial. Here we utilized both RNA and DNA sequence data from high-grade gliomas from the Cancer Genome Atlas to assess the veracity of the association between GBM and HCMV (or other infectious agents). Raw sequence reads were aligned to a reference genome containing a human genome (hg19) and a library of virus sequences known to infect humans. Alignments were performed using Spliced Transcripts Alignments to a Reference (STAR). In 56 out of 56 GBM RNA-seq datasets no known viruses were identified. Virome analysis of the DNA-seq datasets demonstrated 40 out of 40 datasets with human herpesvirus 7 ranging from 32 to 10,845 reads. However, upon further analysis, it was determined that these herpesvirus 7 matching reads have low complexity and are consistent with mapping to human telomeric repeats. Therefore, no known viruses were identified at the DNA or RNA levels. This analysis raises the possibility that viruses may not be associated with gliomas as previously reported.
PMCID: PMC4218198
5.  Ataxin-2 as potential disease modifier in C9ORF72 expansion carriers 
Neurobiology of aging  2014;35(10):2421.e13-2421.e17.
Repeat expansions in chromosome 9 open reading frame 72 (C9ORF72) are an important cause of both motor neuron disease (MND) and frontotemporal dementia (FTD). Currently, little is known about factors that could account for the phenotypic heterogeneity detected in C9ORF72 expansion carriers. In this study, we investigated four genes that could represent genetic modifiers: ataxin-2 (ATXN2), non-imprinted in Prader-Willi/Angelman syndrome 1 (NIPA1), survival motor neuron 1 (SMN1) and survival motor neuron 2 (SMN2). Assessment of these genes, in a unique cohort of 331 C9ORF72 expansion carriers and 376 controls, revealed that intermediate repeat lengths in ATXN2 possibly act as disease modifier in C9ORF72 expansion carriers; no evidence was provided for a potential role of NIPA1, SMN1 or SMN2. The effects of intermediate ATXN2 repeats were most profound in probands with MND or FTD/MND (2.1% versus 0% in controls, P=0.013), whereas the frequency in probands with FTD was identical to controls. Though intermediate ATXN2 repeats were already known to be associated with MND risk, previous reports did not focus on individuals with clear pathogenic mutations, such as repeat expansions in C9ORF72. Based on our present findings, we postulate that intermediate ATXN2 repeat lengths may render C9ORF72 expansion carriers more susceptible to the development of MND; further studies are needed, however, to validate our findings.
PMCID: PMC4105839  PMID: 24866401
C9ORF72; ataxin-2; ATXN2; motor neuron disease; amyotrophic lateral sclerosis; frontotemporal dementia; disease modifier
6.  High-Throughput RNA Sequencing-Based Virome Analysis of 50 Lymphoma Cell Lines from the Cancer Cell Line Encyclopedia Project 
Journal of Virology  2014;89(1):713-729.
Using high-throughput RNA sequencing data from 50 common lymphoma cell culture models from the Cancer Cell Line Encyclopedia project, we performed an unbiased global interrogation for the presence of a panel of 740 viruses and strains known to infect human and other mammalian cells. This led to the findings of previously identified infections by Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV), and human T-lymphotropic virus type 1 (HTLV-1). In addition, we also found a previously unreported infection of one cell line (DEL) with a murine leukemia virus. High expression of murine leukemia virus (MuLV) transcripts was observed in DEL cells, and we identified four transcriptionally active integration sites, one being in the TNFRSF6B gene. We also found low levels of MuLV reads in a number of other cell lines and provided evidence suggesting cross-contamination during sequencing. Analysis of HTLV-1 integrations in two cell lines, HuT 102 and MJ, identified 14 and 66 transcriptionally active integration sites with potentially activating integrations in immune regulatory genes, including interleukin-15 (IL-15), IL-6ST, STAT5B, HIVEP1, and IL-9R. Although KSHV and EBV do not typically integrate into the genome, we investigated a previously identified integration of EBV into the BACH2 locus in Raji cells. This analysis identified a BACH2 disruption mechanism involving splice donor sequestration. Through viral gene expression analysis, we detected expression of stable intronic RNAs from the EBV BamHI W repeats that may be part of long transcripts spanning the repeat region. We also observed transcripts at the EBV vIL-10 locus exclusively in the Hodgkin's lymphoma cell line, Hs 611.T, the expression of which were uncoupled from other lytic genes. Assessment of the KSHV viral transcriptome in BCP-1 cells showed expression of the viral immune regulators, K2/vIL-6, K4/vIL-8-like vCCL1, and K5/E2-ubiquitin ligase 1 that was significantly higher than expression of the latency-associated nuclear antigen. Together, this investigation sheds light into the virus composition across these lymphoma model systems and provides insights into common viral mechanistic principles.
IMPORTANCE Viruses cause cancer in humans. In lymphomas the Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV) and human T-lymphotropic virus type 1 are major contributors to oncogenesis. We assessed virus-host interactions using a high throughput sequencing method that facilitates the discovery of new virus-host associations and the investigation into how the viruses alter their host environment. We found a previously unknown murine leukemia virus infection in one cell line. We identified cellular genes, including cytokine regulators, that are disrupted by virus integration, and we determined mechanisms through which virus integration causes deregulation of cellular gene expression. Investigation into the KSHV transcriptome in the BCP-1 cell line revealed high-level expression of immune signaling genes. EBV transcriptome analysis showed expression of vIL-10 transcripts in a Hodgkin's lymphoma that was uncoupled from lytic genes. These findings illustrate unique mechanisms of viral gene regulation and to the importance of virus-mediated host immune signaling in lymphomas.
PMCID: PMC4301145  PMID: 25355872
7.  Benign Sacral Metastatic Meningioma: A Rare Entity 
The Ochsner Journal  2015;15(2):200-202.
Meningiomas are common intracranial tumors with a low metastatic rate. Those that do metastasize often show histopathologic signs of malignancy. In rare cases, the primary and secondary tumors are histologically benign.
Case Report
We report the case of a 57-year-old female with a histologically benign intracranial meningioma that metastasized to the sacrum. The patient had a long history of intracranial meningioma with multiple recurrences. At each recurrence, histopathologic examination of the resected tumor showed no signs of malignancy. The sacral meningioma was biopsied and found to be histologically benign. The patient was treated with radiotherapy (54 Gy in 30 fractions), and her symptoms resolved. Six months later, the patient developed left leg weakness. Magnetic resonance imaging showed growth of her intracranial mass for which she underwent a craniotomy for tumor resection. Pathologic evaluation showed evidence of benign meningioma without atypical features. She recovered well from this procedure and returned to her baseline in several weeks.
After treatment, the patient had no signs of radiographic progression in either location.
PMCID: PMC4482567  PMID: 26130988
Meningioma; neoplasm metastasis; radiosurgery; sacrum; spine
8.  The emerging role of guanine nucleotide exchange factors in ALS and other neurodegenerative diseases 
Small GTPases participate in a broad range of cellular processes such as proliferation, differentiation, and migration. The exchange of GDP for GTP resulting in the activation of these GTPases is catalyzed by a group of enzymes called guanine nucleotide exchange factors (GEFs), of which two classes: Dbl-related exchange factors and the more recently described dedicator of cytokinesis proteins family exchange factors. Increasingly, deregulation of normal GEF activity or function has been associated with a broad range of disease states, including neurodegeneration and neurodevelopmental disorders. In this review, we examine this evidence with special emphasis on the novel role of Rho guanine nucleotide exchange factor (RGNEF/p190RhoGEF) in the pathogenesis of amyotrophic lateral sclerosis. RGNEF is the first neurodegeneration-linked GEF that regulates not only RhoA GTPase activation but also functions as an RNA binding protein that directly acts with low molecular weight neurofilament mRNA 3′ untranslated region to regulate its stability. This dual role for RGNEF, coupled with the increasing understanding of the key role for GEFs in modulating the GTPase function in cell survival suggests a prominent role for GEFs in mediating a critical balance between cytotoxicity and neuroprotection which, when disturbed, contributes to neuronal loss.
PMCID: PMC4159981  PMID: 25309324
neurofilament; RNA binding proteins; GEF; neurodegeneration; motor neuron disease
9.  TMEM106B protects C9ORF72 expansion carriers against frontotemporal dementia 
Acta neuropathologica  2014;127(3):397-406.
Variants in transmembrane protein 106 B (TMEM106B) modify the disease penetrance of frontotemporal dementia (FTD) in carriers of progranulin (GRN) mutations. We investigated whether TMEM106B is also a genetic modifier of disease in carriers of chromosome 9 open reading frame 72 (C9ORF72) expansions. We assessed the genotype of 325 C9ORF72 expansion carriers (cohort 1), 586 FTD patients lacking C9ORF72 expansions (with or without motor neuron disease [MND]; cohort 2), and a total of 1,302 controls for TMEM106B variants (rs3173615 and rs1990622) using MassArray iPLEX and Taqman genotyping assays. For our primary analysis, we focused on functional variant rs3173615, and employed a recessive genotypic model. In cohort 1, patients with C9ORF72 expansions showed a significantly reduced frequency of carriers homozygous for the minor allele as compared to controls (11.9% versus 19.1%, odds ratio (OR): 0.57, p=0.014; same direction as carriers of GRN mutations). The strongest evidence was provided by FTD patients (OR: 0.33, p=0.009) followed by FTD/MND patients (OR: 0.38, p=0.017), whereas no significant difference was observed in MND patients (OR: 0.85, p=0.55). In cohort 2, the frequency of carriers homozygous for the minor allele was not significantly reduced in patients as compared to controls (OR: 0.77, p=0.079); however, a significant reduction was observed when focusing on those patients with frontotemporal lobar degeneration and TAR DNA-binding protein 43 inclusions (FTLD-TDP; OR: 0.26, p<0.001).
Our study identifies TMEM106B as the first genetic factor modifying disease presentation in C9ORF72 expansion carriers. Homozygosity for the minor allele protects carriers from developing FTD, but not from developing MND; similar effects are seen in FTLD-TDP patients with yet unknown genetic causes. These new findings show that the protective effects of TMEM106B are not confined to carriers of GRN mutations, and might be relevant for prognostic testing, and as a promising therapeutic target for the entire spectrum of FTLD-TDP.
PMCID: PMC3944829  PMID: 24385136
C9ORF72; TMEM106B; frontotemporal dementia; motor neuron disease; amyotrophic lateral sclerosis; disease modifier
10.  Comprehensive High-Throughput RNA Sequencing Analysis Reveals Contamination of Multiple Nasopharyngeal Carcinoma Cell Lines with HeLa Cell Genomes 
Journal of Virology  2014;88(18):10696-10704.
In an attempt to explore infectious agents associated with nasopharyngeal carcinomas (NPCs), we employed our high-throughput RNA sequencing (RNA-seq) analysis pipeline, RNA CoMPASS, to investigate the presence of ectopic organisms within a number of NPC cell lines commonly used by NPC and Epstein-Barr virus (EBV) researchers. Sequencing data sets from both CNE1 and HONE1 were found to contain reads for human papillomavirus 18 (HPV-18). Subsequent real-time reverse transcription-PCR (RT-PCR) analysis on a panel of NPC cell lines identified HPV-18 in CNE1 and HONE1 as well as three additional NPC cell lines (CNE2, AdAH, and NPC-KT). Further analysis of the chromosomal integration arrangement of HPV-18 in NPCs revealed patterns identical to those observed in HeLa cells. Clustering based on human single nucleotide variation (SNV) analysis of two separate HeLa cell lines and several NPC cell lines demonstrated two distinct clusters with CNE1, as well as HONE1 clustering with the two HeLa cell lines. In addition, duplex-PCR-based genotyping showed that CNE1, CNE2, and HONE1 do not have a HeLa cell-specific L1 retrotransposon insertion, suggesting that these three HPV-18+ NPC lines are likely products of a somatic hybridization with HeLa cells, which is also consistent with our RNA-seq-based gene level SNV analysis. Taking all of these findings together, we conclude that a widespread HeLa contamination may exist in many NPC cell lines, and authentication of these cell lines is recommended. Finally, we provide a proof of concept for the utility of an RNA-seq-based approach for cell authentication.
IMPORTANCE Nasopharyngeal carcinoma (NPC) cell lines are important model systems for analyzing the complex life cycle and pathogenesis of Epstein-Barr virus (EBV). Using an RNA-seq-based approach, we found HeLa cell contamination in several NPC cell lines that are commonly used in the EBV and related fields. Our data support the notion that contamination resulted from somatic hybridization with HeLa cells, likely occurring at the point of cell line establishment. Given the rarity of NPCs, the long history of NPC cell lines, and the lack of rigorous cell line authentication, it is likely that the actual prevalence and impact of HeLa cell contamination on the EBV field might be greater. We therefore recommend cell line authentication prior to performing experiments using NPC cell lines to avoid inaccurate conclusions. The novel RNA-seq-based cell authentication approach reported here can serve as a comprehensive method for validating cell lines.
PMCID: PMC4178894  PMID: 24991015
11.  Effects of the Endocrine-Disrupting Chemical DDT on Self-Renewal and Differentiation of Human Mesenchymal Stem Cells 
Background: Although the global use of the endocrine-disrupting chemical DDT has decreased, its persistence in the environment has resulted in continued human exposure. Accumulating evidence suggests that DDT exposure has long-term adverse effects on development, yet the impact on growth and differentiation of adult stem cells remains unclear.
Objectives: Human mesenchymal stem cells (MSCs) exposed to DDT were used to evaluate the impact on stem cell biology.
Methods: We assessed DDT-treated MSCs for self-renewal, proliferation, and differentiation potential. Whole genome RNA sequencing was performed to assess gene expression in DDT-treated MSCs.
Results: MSCs exposed to DDT formed fewer colonies, suggesting a reduction in self-renewal potential. DDT enhanced both adipogenic and osteogenic differentiation, which was confirmed by increased mRNA expression of glucose transporter type 4 (GLUT4), lipoprotein lipase (LpL), peroxisome proliferator-activated receptor gamma (PPARγ), leptin, osteonectin, core binding factor 1 (CBFA1), and FBJ murine osteosarcoma viral oncogene homolog (c-Fos). Expression of factors in DDT-treated cells was similar to that in estrogen-treated MSCs, suggesting that DDT may function via the estrogen receptor (ER)-mediated pathway. The coadministration of ICI 182,780 blocked the effects of DDT. RNA sequencing revealed 121 genes and noncoding RNAs to be differentially expressed in DDT-treated MSCs compared with controls cells.
Conclusion: Human MSCs provide a powerful biological system to investigate and identify the molecular mechanisms underlying the effects of environmental agents on stem cells and human health. MSCs exposed to DDT demonstrated profound alterations in self-renewal, proliferation, differentiation, and gene expression, which may partially explain the homeostatic imbalance and increased cancer incidence among those exposed to long-term EDCs.
Citation: Strong AL, Shi Z, Strong MJ, Miller DF, Rusch DB, Buechlein AM, Flemington EK, McLachlan JA, Nephew KP, Burow ME, Bunnell BA. 2015. Effects of the endocrine-disrupting chemical DDT on self-renewal and differentiation of human mesenchymal stem cells. Environ Health Perspect 123:42–48;
PMCID: PMC4286277  PMID: 25014179
12.  Microbial Contamination in Next Generation Sequencing: Implications for Sequence-Based Analysis of Clinical Samples 
PLoS Pathogens  2014;10(11):e1004437.
The high level of accuracy and sensitivity of next generation sequencing for quantifying genetic material across organismal boundaries gives it tremendous potential for pathogen discovery and diagnosis in human disease. Despite this promise, substantial bacterial contamination is routinely found in existing human-derived RNA-seq datasets that likely arises from environmental sources. This raises the need for stringent sequencing and analysis protocols for studies investigating sequence-based microbial signatures in clinical samples.
PMCID: PMC4239086  PMID: 25412476
14.  Genetic modifiers in carriers of repeat expansions in the C9ORF72 gene 
Hexanucleotide repeat expansions in chromosome 9 open reading frame 72 (C9ORF72) are causative for frontotemporal dementia (FTD) and motor neuron disease (MND). Substantial phenotypic heterogeneity has been described in patients with these expansions. We set out to identify genetic modifiers of disease risk, age at onset, and survival after onset that may contribute to this clinical variability.
We examined a cohort of 330 C9ORF72 expansion carriers and 374 controls. In these individuals, we assessed variants previously implicated in FTD and/or MND; 36 variants were included in our analysis. After adjustment for multiple testing, our analysis revealed three variants significantly associated with age at onset (rs7018487 [UBAP1; p-value = 0.003], rs6052771 [PRNP; p-value = 0.003], and rs7403881 [MT-Ie; p-value = 0.003]), and six variants significantly associated with survival after onset (rs5848 [GRN; p-value = 0.001], rs7403881 [MT-Ie; p-value = 0.001], rs13268953 [ELP3; p-value = 0.003], the epsilon 4 allele [APOE; p-value = 0.004], rs12608932 [UNC13A; p-value = 0.003], and rs1800435 [ALAD; p-value = 0.003]).
Variants identified through this study were previously reported to be involved in FTD and/or MND, but we are the first to describe their effects as potential disease modifiers in the presence of a clear pathogenic mutation (i.e. C9ORF72 repeat expansion). Although validation of our findings is necessary, these variants highlight the importance of protein degradation, antioxidant defense and RNA-processing pathways, and additionally, they are promising targets for the development of therapeutic strategies and prognostic tests.
Electronic supplementary material
The online version of this article (doi:10.1186/1750-1326-9-38) contains supplementary material, which is available to authorized users.
PMCID: PMC4190282  PMID: 25239657
C9ORF72; Frontotemporal dementia; Motor neuron disease; Genetic modifier; Repeat expansion
15.  Comprehensive Luciferase-Based Reporter Gene Assay Reveals Previously Masked Up-Regulatory Effects of miRNAs 
MicroRNAs (miRNAs) are small non-coding RNAs that regulate the majority of the transcriptome at a post-transcriptional level. Because of this critical role, it is important to ensure that the assays used to determine their functionality are robust and reproducible. Typically, the reporter gene assay in cell-based systems has been the first-line method to study miRNA functionality. In order to overcome some of the potential errors in interpretation that can be associated with this assay, we have developed a detailed protocol for the luciferase reporter gene assay that has been modified for miRNAs. We demonstrate that normalization against the effect of the miRNA and cellular factors on the luciferase coding sequence is essential to obtain the specific impact of the miRNA on the 3'UTR (untranslated region) target. Our findings suggest that there is a real possibility that the roles for miRNA in transcriptome regulation may be misreported due to inaccurate normalization of experimental data and also that up-regulatory effects of miRNAs are not uncommon in cells. We propose to establish this comprehensive method as standard for miRNA luciferase reporter assays to avoid errors and misinterpretations in the functionality of miRNAs.
PMCID: PMC4200788  PMID: 25192285
reporter gene assay; miRNA; normalization; NEFL (neurofilament)
16.  Global Bidirectional Transcription of the Epstein-Barr Virus Genome during Reactivation 
Journal of Virology  2014;88(3):1604-1616.
Epstein-Barr virus (EBV) reactivation involves the ordered induction of approximately 90 viral genes that participate in the generation of infectious virions. Using strand-specific RNA-seq to assess the EBV transcriptome during reactivation, we found extensive bidirectional transcription extending across nearly the entire genome. In contrast, only 4% of the EBV genome is currently bidirectionally annotated. Most of the newly identified transcribed regions show little evidence of coding potential, supporting noncoding roles for most of these RNAs. Based on previous cellular long noncoding RNA size calculations, we estimate that there are likely hundreds more EBV genes expressed during reactivation than was previously known. Limited 5′ and 3′ rapid amplification of cDNA ends (RACE) experiments and findings of novel splicing events by RNA-seq suggest that the complexity of the viral genome during reactivation may be even greater. Further analysis of antisense transcripts at some of the EBV latency gene loci showed that they are “late” genes, they are nuclear, and they tend to localize in areas of the nucleus where others find newly synthesized viral genomes. This raises the possibility that these transcripts perform functions such as new genome processing, stabilization, organization, etc. The finding of a significantly more complex EBV transcriptome during reactivation changes our view of the viral production process from one that is facilitated and regulated almost entirely by previously identified viral proteins to a process that also involves the contribution of a wide array of virus encoded noncoding RNAs.
PMCID: PMC3911580  PMID: 24257595
17.  Epstein-Barr Virus and Human Herpesvirus 6 Detection in a Non-Hodgkin's Diffuse Large B-Cell Lymphoma Cohort by Using RNA Sequencing 
Journal of Virology  2013;87(23):13059-13062.
Comprehensive virome analysis of RNA sequence (RNA-seq) data sets from 118 non-Hodgkin's B-cell lymphomas revealed a small subset that is positive for Epstein-Barr virus (EBV) or human herpesvirus 6B (HHV-6B), with one coinfection. EBV transcriptome analysis revealed expression of the latency genes RPMS1, LMP1, and LMP2, with one sample additionally showing a high level of early lytic expression and another sample showing a high level of EBNA2 expression. HHV-6B transcriptome analysis revealed that the majority of genes were transcribed.
PMCID: PMC3838131  PMID: 24049168
18.  RNA CoMPASS: A Dual Approach for Pathogen and Host Transcriptome Analysis of RNA-Seq Datasets 
PLoS ONE  2014;9(2):e89445.
High-throughput RNA sequencing (RNA-seq) has become an instrumental assay for the analysis of multiple aspects of an organism's transcriptome. Further, the analysis of a biological specimen's associated microbiome can also be performed using RNA-seq data and this application is gaining interest in the scientific community. There are many existing bioinformatics tools designed for analysis and visualization of transcriptome data. Despite the availability of an array of next generation sequencing (NGS) analysis tools, the analysis of RNA-seq data sets poses a challenge for many biomedical researchers who are not familiar with command-line tools. Here we present RNA CoMPASS, a comprehensive RNA-seq analysis pipeline for the simultaneous analysis of transcriptomes and metatranscriptomes from diverse biological specimens. RNA CoMPASS leverages existing tools and parallel computing technology to facilitate the analysis of even very large datasets. RNA CoMPASS has a web-based graphical user interface with intrinsic queuing to control a distributed computational pipeline. RNA CoMPASS was evaluated by analyzing RNA-seq data sets from 45 B-cell samples. Twenty-two of these samples were derived from lymphoblastoid cell lines (LCLs) generated by the infection of naïve B-cells with the Epstein Barr virus (EBV), while another 23 samples were derived from Burkitt's lymphomas (BL), some of which arose in part through infection with EBV. Appropriately, RNA CoMPASS identified EBV in all LCLs and in a fraction of the BLs. Cluster analysis of the human transcriptome component of the RNA CoMPASS output clearly separated the BLs (which have a germinal center-like phenotype) from the LCLs (which have a blast-like phenotype) with evidence of activated MYC signaling and lower interferon and NF-kB signaling in the BLs. Together, this analysis illustrates the utility of RNA CoMPASS in the simultaneous analysis of transcriptome and metatranscriptome data. RNA CoMPASS is freely available at
PMCID: PMC3934900  PMID: 24586784
19.  Genetic analysis of SIGMAR1 as a cause of familial ALS with dementia 
Amyotrophic lateral sclerosis (ALS) is the most common motor neuron diseases (MND), while frontotemporal lobar degeneration (FTLD) is the second most common cause of early-onset dementia. Many ALS families segregating FTLD have been reported, particularly over the last decade. Recently, mutations in TARDBP, FUS/TLS, and C9ORF72 have been identified in both ALS and FTLD patients, while mutations in VCP, a FTLD associated gene, have been found in ALS families. Distinct variants located in the 3′-untranslated region (UTR) of the SIGMAR1 gene were previously reported in three unrelated FTLD or FTLD–MND families. We directly sequenced the coding and UTR regions of the SIGMAR1 gene in a targeted cohort of 25 individual familial ALS cases of Caucasian origin with a history of cognitive impairments. This screening identified one variant in the 3′-UTR of the SIGMAR1 gene in one ALS patient, but the same variant was also observed in 1 out of 380 control chromosomes. Subsequently, we screened the same samples for a C9ORF72 repeat expansion: 52% of this cohort was found expanded, including the sample with the SIGMAR1 3′-UTR variant. Consequently, coding and noncoding variants located in the 3′-UTR region of the SIGMAR1 gene are not the cause of FTLD–MND in our cohort, and more than half of this targeted cohort is genetically explained by C9ORF72 repeat expansions.
PMCID: PMC3548267  PMID: 22739338
amyotrophic lateral sclerosis; frontotemporal lobar degeneration; motor neuron disease; dementia
20.  Analysis of Novel NEFL mRNA Targeting microRNAs in Amyotrophic Lateral Sclerosis 
PLoS ONE  2014;9(1):e85653.
Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by progressive motor neuron degeneration and neurofilament aggregate formation. Spinal motor neurons in ALS also show a selective suppression in the levels of low molecular weight neurofilament (NEFL) mRNA. We have been interested in investigating the role of microRNAs (miRNAs) in NEFL transcript stability. MiRNAs are small, 20–25 nucleotide, non-coding RNAs that act as post-transcriptional gene regulators by targeting the 3′ untranslated region (3′UTR) of mRNA resulting in mRNA decay or translational silencing. In this study, we characterized putative novel miRNAs from a small RNA library derived from control and sporadic ALS (sALS) spinal cords. We detected 80 putative novel miRNAs, 24 of which have miRNA response elements (MREs) within the NEFL mRNA 3′UTR. From this group, we determined by real-time PCR that 10 miRNAs were differentially expressed in sALS compared to controls. Functional analysis by reporter gene assay and relative quantitative RT-PCR showed that two novel miRNAs, miR-b1336 and miR-b2403, were downregulated in ALS spinal cord and that both stabilize NEFL mRNA. We confirmed the direct effect of these latter miRNAs using anit-miR-b1336 and anti-miR-b2403. These results demonstrate that the expression of two miRNAs (miRNAs miR-b1336 and miR-b2403) whose effect is to stabilize NEFL mRNA are down regulated in ALS, the net effect of which is predicted to contribute directly to the loss of NEFL steady state mRNA which is pathognomic of spinal motor neurons in ALS.
PMCID: PMC3893244  PMID: 24454911
21.  Prognosis of amyotrophic lateral sclerosis with respiratory onset 
Respiratory muscle involvement is a recognised, but often late, complication of amyotrophic lateral sclerosis (ALS). The clinical features and prognosis of 21 patients with respiratory onset ALS are reported here. On a retrospective chart review, it was found that 2.7% of patients with ALS presenting to a tertiary care specialty clinic have respiratory symptoms as their first clinical symptom of ALS. Only 14% of these individuals presented acutely and required emergency intubation. The mean survival time of the total group from symptom onset to death or permanent ventilation was 27.0 (14.9) months, which was not significantly different from the survival time in patients with bulbar onset ALS. Non‐invasive positive pressure ventilation (NIPPV) significantly improved survival compared with those who did not use NIPPV. This study suggests that ALS with respiratory onset does not necessarily follow a rapidly progressive course.
PMCID: PMC2077959  PMID: 17088331
22.  Length of normal alleles of C9ORF72 GGGGCC repeat do not influence disease phenotype 
Neurobiology of aging  2012;33(12):2950.e5-2950.e7.
Expansions of the non-coding GGGGCC hexanucleotide repeat in the chromosome 9 open reading frame 72 (C9ORF72) gene were recently identified as the long sought-after cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) on chromosome 9p. In this study we aimed to determine whether the length of the normal - unexpanded - allele of the GGGGCC repeat in C9ORF72 plays a role in the presentation of disease or affects age at onset in C9ORF72 mutation carriers. We also studied whether the GGGGCC repeat length confers risk or affects age at onset in FTD and ALS patients without C9ORF72 repeat expansions. C9ORF72 genotyping was performed in 580 FTD, 995 ALS and 160 FTD-ALS patients and 1444 controls, leading to the identification of 211 patients with pathogenic C9ORF72 repeat expansions and an accurate quantification of the length of the normal alleles in all patients and controls. No meaningful association between the repeat length of the normal alleles of the GGGGCC repeat in C9ORF72 and disease phenotype or age at onset was observed in C9ORF72 mutation carriers or non-mutation carriers.
PMCID: PMC3617405  PMID: 22840558
Amyotrophic lateral sclerosis; Frontotemporal Dementia; C9ORF72; Repeat-expansion disease; Association study
23.  Whole-Genome Sequencing of the Akata and Mutu Epstein-Barr Virus Strains 
Journal of Virology  2013;87(2):1172-1182.
Using a simple viral genome enrichment approach, we report the de novo assembly of the Akata and Mutu Epstein-Barr virus (EBV) genomes from a single lane of next-generation sequencing (NGS) reads. The Akata and Mutu viral genomes are type I EBV strains of approximately 171 kb in length. Evidence for genome heterogeneity was found for the Akata but not for the Mutu strain. A comparative analysis of Akata with another four completely sequenced EBV strains, B95-8/Raji, AG876, Mutu, and GD1, demonstrated that the Akata strain is most closely related to the GD1 strain and exhibits the greatest divergence from the type II strain, AG876. A global comparison of latent and lytic gene sequences showed that the four latency genes, EBNA2, EBNA3A, EBNA3B, and EBNA3C, are uniquely defining of type I and type II strain differences. Within type I strains, LMP1, the latency gene, is among the most divergent of all EBV genes, with three insertion or deletion loci in its CTAR2 and CTAR3 signaling domains. Analysis of the BHLF1 and LF3 genes showed that the reading frames identified in the B95-8/Raji genome are not conserved in Akata (or Mutu, for BHLF1), suggesting a primarily non-protein-coding function in EBV's life cycle. The Akata and Mutu viral-genome sequences should be a useful resource for homology-based functional prediction and for molecular studies, such as PCR, RNA-seq, recombineering, and transcriptome studies. As an illustration, we identified novel RNA-editing events in ebv-miR-BART6 antisense transcripts using the Akata and Mutu reference genomes.
PMCID: PMC3554088  PMID: 23152513
24.  Altered microRNA expression profile in amyotrophic lateral sclerosis: a role in the regulation of NFL mRNA levels 
Molecular Brain  2013;6:26.
Amyotrophic Lateral Sclerosis (ALS) is a progressive, adult onset, fatal neurodegenerative disease of motor neurons. There is emerging evidence that alterations in RNA metabolism may be critical in the pathogenesis of ALS. MicroRNAs (miRNAs) are small non-coding RNAs that are key determinants of mRNA stability. Considering that miRNAs are increasingly being recognized as having a role in a variety of neurodegenerative diseases, we decided to characterize the miRNA expression profile in spinal cord (SC) tissue in sporadic ALS (sALS) and controls. Furthermore, we performed functional analysis to identify a group of dysregulated miRNAs that could be responsible for the selective suppression of low molecular weight neurofilament (NFL) mRNA observed in ALS.
Using TaqMan arrays we analyzed 664 miRNAs and found that a large number of miRNAs are differentially expressed in ventral lumbar SC in sALS compared to controls. We observed that the majority of dysregulated miRNAs are down-regulated in sALS SC tissues. Ingenuity Pathway Analysis (IPA) showed that dysregulated miRNAs are linked with nervous system function and cell death. We used two prediction algorithms to develop a panel of miRNAs that have recognition elements within the human NFL mRNA 3′UTR, and then we performed functional analysis for these miRNAs. Our results demonstrate that three miRNAs that are dysregulated in sALS (miR-146a*, miR-524-5p and miR-582-3p) are capable of interacting with NFL mRNA 3′UTR in a manner that is consistent with the suppressed steady state mRNA levels observed in spinal motor neurons in ALS.
The miRNA expression profile is broadly altered in the SC in sALS. Amongst these is a group of dysregulated miRNAs directly regulate the NFL mRNA 3′UTR, suggesting a role in the selective suppression of NFL mRNA in the ALS spinal motor neuron neurofilamentous aggregate formation.
PMCID: PMC3668997  PMID: 23705811
miRNA; Amyotrophic lateral sclerosis; Neurofilament
25.  Differences in Gastric Carcinoma Microenvironment Stratify According to EBV Infection Intensity: Implications for Possible Immune Adjuvant Therapy 
PLoS Pathogens  2013;9(5):e1003341.
Epstein-Barr virus (EBV) is associated with roughly 10% of gastric carcinomas worldwide (EBVaGC). Although previous investigations provide a strong link between EBV and gastric carcinomas, these studies were performed using selected EBV gene probes. Using a cohort of gastric carcinoma RNA-seq data sets from The Cancer Genome Atlas (TCGA), we performed a quantitative and global assessment of EBV gene expression in gastric carcinomas and assessed EBV associated cellular pathway alterations. EBV transcripts were detected in 17% of samples but these samples varied significantly in EBV coverage depth. In four samples with the highest EBV coverage (hiEBVaGC – high EBV associated gastric carcinoma), transcripts from the BamHI A region comprised the majority of EBV reads. Expression of LMP2, and to a lesser extent, LMP1 were also observed as was evidence of abortive lytic replication. Analysis of cellular gene expression indicated significant immune cell infiltration and a predominant IFNG response in samples expressing high levels of EBV transcripts relative to samples expressing low or no EBV transcripts. Despite the apparent immune cell infiltration, high levels of the cytotoxic T-cell (CTL) and natural killer (NK) cell inhibitor, IDO1, was observed in the hiEBVaGCs samples suggesting an active tolerance inducing pathway in this subgroup. These results were confirmed in a separate cohort of 21 Vietnamese gastric carcinoma samples using qRT-PCR and on tissue samples using in situ hybridization and immunohistochemistry. Lastly, a panel of tumor suppressors and candidate oncogenes were expressed at lower levels in hiEBVaGC versus EBV-low and EBV-negative gastric cancers suggesting the direct regulation of tumor pathways by EBV.
Author Summary
Epstein-Barr virus (EBV) is detected in roughly 10% of gastric carcinoma (GC) cases worldwide. Despite a strong link between EBV and gastric carcinoma, the contribution of EBV to the tumor environment in EBV associated gastric carcinoma is unclear. We performed a global assessment of EBV and host cell gene expression in gastric carcinoma tumors from 71 patients to link EBV genes (and expression intensities) to cell and microenvironmental changes. In addition to the finding that EBV is associated with down-regulated tumor regulatory genes, this study revealed that samples with high levels of EBV gene expression (hiEBVaGCs) displayed elevated immune cell infiltration with high interferon-gamma (IFNG) expression compared to samples with low or no EBV gene expression. Despite this evidence of increased immune posturing, hiEBVaGC samples also showed elevated expression of the potent immune cell inhibitor, IDO1. This finding may partly explain the persistence of these virus associated tumors in the face of local immune cell concentration. Importantly, the small molecule IDO inhibitor, 1MT (1-methyl Tryptophan), has been shown to reverse the tolerance inducing effects of IDO1 in other tumors. We propose that stratification of gastric carcinomas into EBV-negative, EBV-low and EBV-high may provide indicator value for the use of IDO1 inhibitors as adjuvant therapies against hiEBVaGCs.
PMCID: PMC3649992  PMID: 23671415

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