scholarly journals Comprehensive Gene Analysis of IgG4-Related Ophthalmic Disease Using RNA Sequencing

2020 ◽  
Vol 9 (11) ◽  
pp. 3458
Author(s):  
Masaki Asakage ◽  
Yoshihiko Usui ◽  
Naoya Nezu ◽  
Hiroyuki Shimizu ◽  
Kinya Tsubota ◽  
...  
Keyword(s):  
Real Time ◽  
Rna Sequencing ◽  
Malt Lymphoma ◽  
Lymphoid Hyperplasia ◽  
Reactive Lymphoid Hyperplasia ◽  
Rna Seq ◽  
Immunoglobulin G4 ◽  
Tissue Samples ◽  
Biopsy Specimens ◽  
Ophthalmic Disease

High-throughput RNA sequencing (RNA-seq) uses massive parallel sequencing technology, allowing the unbiased analysis of genome-wide transcription levels and tumor mutation status. Immunoglobulin G4-related ophthalmic disease (IgG4-ROD) is a fibroinflammatory disease characterized by the enlargement of the ocular adnexal tissues. We analyzed RNA expression levels via RNA-seq in the biopsy specimens of three patients diagnosed with IgG4-ROD. Mucosa-associated lymphoid tissue (MALT) lymphoma, reactive lymphoid hyperplasia (RLH), normal lacrimal gland tissue, and adjacent adipose tissue were used as the controls (n = 3 each). RNA-seq was performed using the NextSeq 500 system, and genes with |fold change| ≥ 2 and p < 0.05 relative to the controls were defined as differentially expressed genes (DEGs) in IgG4-ROD. To validate the results of RNA-seq, real-time polymerase chain reaction (PCR) was performed in 30 IgG4-ROD and 30 orbital MALT lymphoma tissue samples. RNA-seq identified 35 up-regulated genes, including matrix metallopeptidase 12 (MMP12) and secreted phosphoprotein 1 (SPP1), in IgG4-ROD tissues when compared to all the controls. Many pathways related to the immune system were included when compared to all the controls. Expressions of MMP12 and SPP1 in IgG4-ROD tissues were confirmed by real-time PCR and immunohistochemistry. In conclusion, we identified novel DEGs, including those associated with extracellular matrix degradation, fibrosis, and inflammation, in IgG4-ROD biopsy specimens. These data provide new insights into molecular pathogenetic mechanisms and may contribute to the development of new biomarkers for diagnosis and molecular targeted drugs.

Reactive Lymphoid Hyperplasia or Tubercular Lymphadenitis: Can Real-Time PCR on Fine-Needle Aspirates Help Physicians in Concluding the Diagnosis?

2018 ◽  
Vol 62 (3) ◽  
pp. 204-208 ◽  
Author(s):  
Vivek Gupta ◽  
Arvind Bhake
Keyword(s):  
Lymph Node ◽  
Lymph Nodes ◽  
Real Time ◽  
Predictive Value ◽  
Lymphoid Hyperplasia ◽  
Reactive Lymphoid Hyperplasia ◽  
Rt Pcr ◽  
Likelihood Ratios ◽  
Diagnostic Challenge ◽  
Cross Sectional

Background: Enlarged lymph nodes in adult patients often present a diagnostic challenge. In the absence of granuloma or necrosis, the cytology/tissue findings are misleading and relate the enlarged lymph nodes to reactive lymphoid hyperplasia (RLH), because granuloma formation is an immunological response that usually takes 14–100 days to develop. This study assesses the role of real-time (RT)-PCR in the diagnosis of the Mycobacterium complex (MTBC) in lymph node aspirates compared with culture in cases of RLH. Methods: A cross-sectional study was conducted on 112 patients, aged 15–74 years, with a diagnosis of RLH on cytology. RT-PCR for MTBC detection and culture on Löwenstein-Jensen medium for tubercular bacilli was done on lymph node aspirates. Comparative values with reference to culture were calculated. The χ2 value, positive predictive value (PPV), negative predictive value (NPV), and likelihood ratios (LR) were calculated. Results: Out of 112 RLH cases, 35 (31%) were positive on both RT-PCR and culture. RT-PCR was positive in 43 cases and culture was positive in 44 cases. The χ2 test was found to be highly significant. PPV, NPV, positive LR, and negative LR were 81.4%, 87%, 6.76, and 0.23, respectively. Conclusion: RT-PCR for MTBC proves to be useful in arriving at a conclusive diagnosis in patients with a cytological diagnosis of RLH.

EPCO-33. CELL-TYPE PROPORTION DECONVOLUTION OF PEDIATRIC CENTRAL NERVOUS SYSTEM TUMORS FROM SINGLE NUCLEI RNA-seq UNCOVERS UNDERLYING TRANSCRIPTOMIC CHANGES FROM BULK TUMOR RNA-seq COMPARED TO NORMAL BRAIN

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi9-vi9
Author(s):  
Min Kyung Lee ◽  
Nasim Azizgolshani ◽  
Fred Kolling ◽  
Lananh Nguyen ◽  
George Zanazzi ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Rna Sequencing ◽  
Differentially Expressed ◽  
Normal Brain ◽  
Rna Seq ◽  
Cell Type ◽  
Tissue Samples ◽  
Pediatric Brain ◽  
Bulk Tissue ◽  
Unadjusted Analysis

Abstract Identifying transcriptomic alterations in pediatric central nervous system (pCNS) tumors often relies on transcriptomic profiles from bulk tissue RNA-sequencing that can be confounded by varying cell type proportions across tumor and normal brain tissues. We utilized single nuclei RNA-sequencing (snRNA-seq) and bulk RNA-seq in 33 pCNS tumors and 3 non-diseased pediatric brain tissue samples collected from the Norris Cotton Cancer Center to identify variation in gene expression in bulk tissue attributed to overrepresentation of specific cell-type populations when determining differentially expressed genes comparing pCNS tumors to normal pediatric brain tissues. snRNA-seq of 43,515 nuclei (mean = 1,209 nuclei/sample) revealed large proportions of astrocytes (median = 0.45, range = 0.24–0.93) and oligodendrocytes (median = 0.37, range = 0.00–0.66) in pCNS tumors. Compared to normal pediatric brain, proportions of astrocytes were significantly higher (P = 9.2E-03) and neurons were significantly lower (P = 9.4E-03) in pCNS tumors. Differential expression analyses comparing bulk RNA-sequencing data from pCNS tumors to normal pediatric brain identified 902 additional differentially expressed genes (# DE genes = 1,802) when adjusting for astrocyte and neuron proportions compared with unadjusted analysis (# DE genes = 900). In cell-type proportion unadjusted analysis, top DE genes included astrocyte-specific markers, GFAP and CIITA, both of which were found to be not significantly differentially expressed in cell-type proportion adjusted analysis. Indeed, pathways enrichment analysis revealed DE genes in unadjusted models were associated with processes of the neurons and astrocytes such as interferon signaling and postsynaptic signal transmission. After adjustment for astrocyte and neuron proportions, DE genes were associated with defensins and DNA replication-related processes. Our results highlight new potential biological pathways essential in pCNS tumors and indicate the significance of the distribution of varying cell types in tissue samples when conducting studies to investigate transcriptomic alterations in bulk tissue of pCNS tumors.

scholarly journals A relative comparison between Hidden Markov- and Log-Linear-based models for differential expression analysis in a real time course RNA sequencing data

2018 ◽  
Author(s):  
Fatemeh Gholizadeh ◽  
Zahra Salehi ◽  
Ali Mohammad banaei-Moghaddam ◽  
Abbas Rahimi Foroushani ◽  
Kaveh kavousi
Keyword(s):  
Real Time ◽  
Differential Expression ◽  
Rna Sequencing ◽  
Time Course ◽  
Hidden Markov ◽  
Differential Expression Analysis ◽  
Rna Seq ◽  
Sequencing Data ◽  
Normalization Methods ◽  
Log Linear

AbstractWith the advent of the Next Generation Sequencing technologies, RNA-seq has become known as an optimal approach for studying gene expression profiling. Particularly, time course RNA-seq differential expression analysis has been used in many studies to identify candidate genes. However, applying a statistical method to efficiently identify differentially expressed genes (DEGs) in time course studies is challenging due to inherent characteristics of such data including correlation and dependencies over time. Here we aim to relatively compare EBSeq-HMM, a Hidden Markov-based model, with multiDE, a Log-Linear-based model, in a real time course RNA sequencing data. In order to conduct the comparison, common DEGs detected by edgeR, DESeq2 and Voom (referred to as Benchmark DEGs) were utilized as a measure. Each of the two models were compared using different normalization methods. The findings revealed that multiDE identified more Benchmark DEGs and showed a higher agreement with them than EBSeq-HMM. Furthermore, multiDE and EBSeq-HMM displayed their best performance using TMM and Upper-Quartile normalization methods, respectively.

scholarly journals Distinctive Tissue and Serum MicroRNA Profile of IgG4-Related Ophthalmic Disease and MALT Lymphoma

2020 ◽  
Vol 9 (8) ◽  
pp. 2530
Author(s):  
Naoya Nezu ◽  
Yoshihiko Usui ◽  
Masaki Asakage ◽  
Hiroyuki Shimizu ◽  
Kinya Tsubota ◽  
...  
Keyword(s):  
Malt Lymphoma ◽  
Target Genes ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Therapeutic Interventions ◽  
Immunoglobulin G4 ◽  
Serum Microrna ◽  
Microrna Profile ◽  
Mitogen Activated Protein ◽  
Ophthalmic Disease

The molecular pathogenesis of orbital lymphoproliferative disorders, such as immunoglobulin G4-related ophthalmic disease (IgG4-ROD) and orbital mucosa-associated lymphoid tissue (MALT) lymphoma, remains essentially unknown. Differentiation between the two disorders, which is important since the work-up and treatment can vary greatly, is often challenging due to the lack of specific biomarkers. Although miRNAs play an important role in the regulation of carcinogenesis and inflammation, the relationship between miRNA and orbital lymphoproliferative diseases remains unknown. We performed a comprehensive analysis of 2565 miRNAs from biopsy and serum specimens of 17 cases with IgG4-ROD, where 21 cases with orbital MALT lymphoma were performed. We identified specific miRNA signatures and their miRNA target pathways, as well as the network analysis for IgG4-ROD and orbital MALT lymphoma. Machine-learning analysis identified miR-202-3p and miR-7112-3p as the best discriminators of IgG4-ROD and orbital MALT lymphoma, respectively. Enrichment analyses of biological pathways showed that the longevity-regulating pathway in IgG4-ROD and the mitogen-activated protein kinase (MAPK) signaling pathway in orbital MALT lymphoma was most enriched by target genes of downregulated miRNAs. This is the first evidence of miRNA profiles of biopsy and serum specimens of patients with IgG4-ROD and orbital MALT lymphoma. These data will be useful for developing diagnostic and therapeutic interventions, as well as elucidating the pathogenesis of these disorders.

Assessment of Clinically Suspected Tubercular Lymphadenopathy by Real-Time PCR Compared to Non-Molecular Methods on Lymph Node Aspirates

2017 ◽  
Vol 62 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Vivek Gupta ◽  
Arvind Bhake
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Cross Sectional Study ◽  
Lymphoid Hyperplasia ◽  
Reactive Lymphoid Hyperplasia ◽  
Diagnostic Modality ◽  
Cross Sectional ◽  
Microbiological Methods ◽  
Necrotizing Granuloma ◽  
Tubercular Lymphadenitis

Background: The diagnosis of tubercular lymphadenitis (TBLN) is challenging. This study assesses the role of diagnostic intervention with real-time PCR in clinically suspected tubercular lymphadenopathy in relation to cytology and microbiological methods. Methods: The cross-sectional study involved 214 patients, and PCR, cytology, and Ziehl-Neelsen (ZN) staining was performed on aspirates. The findings were compared with culture on Lowenstein-Jensen medium. The overall concordance of cytology and PCR, both individually and combined, was calculated. χ2 and Phi values were assessed between cytology, PCR, and culture. Results: A cytological diagnosis of tuberculosis (TB), reactive lymphoid hyperplasia, and suppurative lymphadenitis was made in 71, 112, and 6 patients, respectively. PCR and culture were positive in 40% of the cases. Among the TBLN patients, PCR showed higher positivity in necrosis and culture showed higher positivity in necrotizing granuloma. Positive ZN staining was observed in 29.6% of the TBLN cases, with an overall positivity of 11%. PCR could additionally detect 82 cases missed by ZN staining. The overall concordance rate for either diagnostic modality, i.e., PCR or cytology, was highest (75%), and for PCR alone was 74%. Phi values were observed to be 0.47 between PCR and culture. Conclusion: Real-time PCR for Mycobacterium tuberculosis complex on aspirates offers a definitive and comparable diagnosis of TBLN. Including this approach as the primary investigation in the work-up of TBLN could reduce the burden of TB.

Molecular Diagnosis of Tubercular Lymphadenopathy from Fine-Needle Aspirates in Pediatric Patients

2017 ◽  
Vol 61 (3) ◽  
pp. 173-178 ◽  
Author(s):  
Vivek Gupta ◽  
Arvind Bhake
Keyword(s):  
Real Time ◽  
Sensitivity And Specificity ◽  
Real Time Pcr ◽  
Pediatric Patients ◽  
Strong Association ◽  
Immunological Response ◽  
Lymphoid Hyperplasia ◽  
Reactive Lymphoid Hyperplasia ◽  
Cross Sectional ◽  
Fine Needle

Objectives: The diagnosis of peripheral tubercular lymphadenopathy (TBLN) in pediatric patients is often a challenge because features evident on fine-needle aspiration cytology (FNAC) or tissue biopsy can be deceptive for the reason that they result from an immunological response. This study aimed to evaluate polymerase chain reaction (PCR) for Mycobacterium tuberculosis complex (MTBC) in pediatric patients under clinical suspicion for TBLN and to assess its role in the evaluation of cases cytodiagnosed as reactive lymphoid hyperplasia. Methods: This was a cross-sectional study conducted on 45 pediatric patients clinically suspected and unsuspected for TBLN. FNAC, culture on Löwenstein-Jensen medium, and real-time PCR were performed. Comparative values with reference to the culture were calculated. Results: Cytology had a sensitivity and specificity of 38.5 and 87.5%, respectively. Real-time PCR had a sensitivity and specificity of 84.6 and 81.3%, respectively. Of the 32 cases with a cytodiagnosis of reactive lymphoid hyperplasia, 53% were positive both on PCR and culture for M. tuberculosis; the φ value of 0.93 demonstrated a strong association between these 2 methods. Conclusion: Real-time PCR is useful in detecting MTBC in pediatric patients, and it also helps in the diagnosis of cases missed on FNAC.

scholarly journals RNA-sequencing analysis of differential gene expression associated with arterial stiffness

Vascular ◽  
2020 ◽  
Vol 28 (5) ◽  
pp. 655-663 ◽  
Author(s):  
Jeongok G Logan ◽  
Sijung Yun ◽  
Yongde Bao ◽  
Emily Farber ◽  
Charles R Farber
Keyword(s):  
Gene Expression ◽  
Cardiovascular Disease ◽  
Arterial Stiffness ◽  
Real Time ◽  
Rna Sequencing ◽  
Real Time Pcr ◽  
Disease Risk ◽  
Expression Profiles ◽  
Sequencing Analysis ◽  
Rna Seq

Objectives Arterial stiffness is recognized as an important predictor of cardiovascular disease morbidity and mortality, independent of traditional cardiovascular disease risk factors. Given that arterial tissue is not easily accessible, most gene expression studies on arterial stiffness have been conducted on animals or on patients who have undergone by-pass surgeries. In order to obtain a deeper understanding of early changes of arterial stiffness, this study compared transcriptome profiles between healthy adults with higher and lower arterial stiffness. Methods The sample included 20 healthy female adults without cardiovascular disease. Arterial stiffness was measured by carotid-femoral pulse wave velocity, the “gold-standard” measure of central arterial stiffness. Peripheral blood samples collected to PAXgene™ RNA tubes were used for RNA sequencing (RNA-seq). The potential confounding effects of age, body mass index, and mean arterial pressure were controlled for in RNA-seq analysis. To validate RNA-seq results, quantitative real-time PCR (qRT-PCR) was performed for six selected genes. Results The findings demonstrated that genes including CAPN9, IL32, ERAP2, RAB6B, MYBPH, and miRNA626 were down-regulated, and that MOCS1 gene was up-regulated among the people with higher arterial stiffness. Real-time PCR showed that the changes of CAPN9, IL32, ERAP2, and RAB6B were in concordance with RNA-seq data, and confirmed the validity of the gene expression profiles obtained by RNA-seq analysis. Conclusions Previous studies have suggested the potential roles of CAPN9, IL32, and ERAP2 in structural changes of the arterial wall through up-regulation of metalloproteinases. However, the current study showed that CAPN9, IL32, and ERAP2 were down-regulated in the individuals with higher arterial stiffness, compared with those with lower arterial stiffness. The unexpected directions of expression of these genes may indicate an effort to maintain vascular homeostasis during increased arterial stiffness among healthy individuals. Further studies are guaranteed to investigate the roles of CAPN9, IL32, and ERAP2 in regulating arterial stiffness in people with and without cardiovascular disease.

Analyzing Molecular Basis of Heat-Induced Loss-of-Wheat Resistance to Hessian Fly (Diptera: Cecidomyiidae) Infestation Using RNA-Sequencing

2020 ◽  
Vol 113 (3) ◽  
pp. 1504-1512
Author(s):  
Lieceng Zhu ◽  
Jiazheng (John) Yuan ◽  
Jordan O’Neal ◽  
Daria Brown ◽  
Ming-Shun Chen
Keyword(s):  
Heat Stress ◽  
Rna Sequencing ◽  
Molecular Basis ◽  
Hessian Fly ◽  
Auxin Signaling ◽  
Primary Metabolism ◽  
Rna Seq ◽  
Tissue Samples ◽  
Expression Of Genes ◽  
The Impact

Abstract Heat stress compromises wheat resistance to Hessian fly (HF, Mayetiola destructor (Say)) (Diptera: Cecidomyiidae) infestation. The objective of this research is to analyze the molecular basis of heat-induced loss of wheat resistance to HF infestation using RNA Sequencing (RNA-seq). To this end, two resistant wheat cultivars ‘Molly’ and ‘Caldwell’ containing the resistance genes H13 and H6, respectively, were infested with an avirulent HF biotype GP and treated with different temperatures to examine the impact of heat stress on their resistance phenotypes. Tissue samples collected from HF feeding sites in Molly plants were subjected to RNA-seq analysis to determine the effect of heat stress on transcript expression of genes in wheat plants. Our results indicate that resistance to HF infestation in Caldwell is more sensitive to heat stress than that in Molly, and that heat stress down-regulates most genes involved in primary metabolism and biosynthesis of lignin and cuticular wax, but up-regulate most or all genes involved in auxin and 12-oxo-phytodienoic acid (OPDA) signaling pathways. Our results and previous reports suggest that heat stress may impair the processes in wheat plants that produce and mobilize chemical resources needed for synthesizing defensive compounds, weaken cell wall and cuticle defense, decrease OPDA signaling, but increase auxin signaling, leading to the suppressed resistance and activation of susceptibility.

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