scholarly journals Exosomal microRNAs are novel circulating biomarkers in cigarette, waterpipe smokers, E-cigarette users and dual smokers

2020 ◽  
Author(s):  
Kameshwar P. Singh ◽  
Krishna P. Maremanda ◽  
Dongmei Li ◽  
Irfan Rahman
Keyword(s):  
Differential Expression ◽  
Health Effects ◽  
Molecular Mechanisms ◽  
Electronic Cigarettes ◽  
Differential Expression Analysis ◽  
Pairwise Comparison ◽  
Gene Set Enrichment Analysis ◽  
Cigarette Smoke Exposure ◽  
Tobacco Exposure ◽  
Non Coding Rnas

Abstract Background Electronic cigarettes (e-cigs) produce aerosolized substances by heating a liquid, which contains large number of chemicals. The aerosol generated by E-cig may produce serious health effects. Cigarette smoke exposure may causes various diseases including COPD, atherosclerosis, and lung cancer. Waterpipe tobacco smoking also causes various acute and chronic health effects including cardiopulmonary diseases. MicroRNAs are present in higher concentration in exosomes that play a major role in various normal physiological functions and diseases. We hypothesized that the non-coding RNAs transcript may serve as susceptibility to disease biomarkers by smoking and vaping. Results Our data show the enrichment of various non-coding RNAs that include microRNAs, tRNAs, piRNAs, snoRNAs, snRNAs, Mt-tRNAs, and other biotypes in exosomes. The detailed differential expression analysis of microRNAs, tRNAs and piRNA showed significant changes between pairwise comparisons of different groups. The common changes in differential expression of 8 microRNAs that are hsa-let-7a-5p, hsa-miR-21-5p, hsa-miR-29b-3p, hsa-let-7f-5p, hsa-miR-143-3p, hsa-miR-30a-5p, hsa-let-7i-5p, and hsa-let-7g-5p were found when compared with all smoking and vaping groups with non-smoking group. The e-cig group has differentially expressed 7 microRNAs (hsa-miR-224-5p, hsa-let-7c-5p, hsa-miR-193b-3p, hsa-miR-30e-5p, hsa-miR-423-3p, hsa-miR-500b-3p, hsa-miR-365a-3p|hsa-miR-365b-3p) that is specific for this group, not expressed in other three groups. Gene set enrichment analysis of microRNA showed significant changes in the top six enriched functions that consisted of biological pathway, biological process, molecular function, cellular component, site of expression and transcription factor in all groups. Further, the pairwise comparison of tRNAs and piRNA in all groups also revealed significant changes in differential expression. Conclusions Plasma exosomes of cigarette smokers, waterpipe smokers, e-cig users and dual smokers have common differential expression of microRNAs (hsa-let-7a-5p, hsa-miR-21-5p, hsa-miR-29b-3p, hsa-let-7f-5p, hsa-miR-143-3p, hsa-miR-30a-5p, hsa-let-7i-5p, and hsa-let-7g-5p), may be biomarker for tobacco exposure. Additionally, the e-cig users have also differential expressed microRNAs (hsa-miR-224-5p, hsa-let-7c-5p, hsa-miR-193b-3p, hsa-miR-30e-5p, hsa-miR-423-3p, hsa-miR-500b-3p, and hsa-miR-365a-3p|hsa-miR-365b-3p) that is specific for this group. This study will help to better understand molecular mechanisms of plasma exosome non-coding RNAs and in developing biomarkers that may be useful in diagnosis and therapy of pulmonary injury and disease by smoking and vaping.

scholarly journals Exosomal microRNAs are novel circulating biomarkers in cigarette,waterpipe smokers, E-cigarette users and dual smokers

2020 ◽  
Author(s):  
Kameshwar P. Singh ◽  
Krishna P. Maremanda ◽  
Dongmei Li ◽  
Irfan Rahman
Keyword(s):  
Differential Expression ◽  
Health Effects ◽  
Tobacco Smoking ◽  
Electronic Cigarettes ◽  
Differential Expression Analysis ◽  
Gene Set Enrichment Analysis ◽  
Differentially Expressed ◽  
Cigarette Smoke Exposure ◽  
Roc Curve Analysis ◽  
Non Coding Rnas

Abstract Background: Electronic cigarettes (e-cigs) produce aerosolized substances by heating a liquid, which contains large number of chemicals. The aerosol generated by E-cig may produce adverse health effects. Cigarette smoke exposure causes various diseases including COPD, atherosclerosis, and lung cancer. Waterpipe tobacco smoking also causes various acute and chronic health effects including cardiopulmonary diseases. microRNAs are present in higher concentration in exosomes that play a major role in various physiological and pathological functions. We hypothesized that the non-coding RNAs transcript may serve as susceptibility to disease biomarkers by smoking and vaping. Results: Our data show the enrichment of various non-coding RNAs that include microRNAs, tRNAs, piRNAs, snoRNAs, snRNAs, Mt-tRNAs, and other biotypes in exosomes. A comprehensive differential expression analysis of microRNAs, tRNAs and piRNAs showed significant changes across different pairwise comparisons. The seven microRNAs that were common and differentially expressed of when all the smoking and vaping groups were compared with non-smokers (NS) are hsa-let-7a-5p, hsa-miR-21-5p, hsa-miR-29b-3p, hsa-let-7f-5p, hsa-miR-143-3p, hsa-miR-30a-5p and hsa-let-7i-5p. The e-cig vs. NS group has differentially expressed 5 microRNAs (hsa-miR-224-5p, hsa-miR-193b-3p, hsa-miR-30e-5p, hsa-miR-423-3p, hsa-miR-365a-3p, and hsa-miR-365b-3p) that is specific for this group and are not expressed in other three groups. Gene set enrichment analysis of microRNAs showed significant changes in the top six enriched functions that consisted of biological pathway, biological process, molecular function, cellular component, site of expression and transcription factor in all the groups. Further, the pairwise comparison of tRNAs and piRNA in all these groups revealed significant changes in their expressions. Conclusions: Plasma exosomes of cigarette smokers, waterpipe smokers, e-cig users and dual smokers have common differential expression of microRNAs which may serve to distinguish smoking and vaping subjects from NS. Among them has-let-7a-5p has high sensitivity and specificity to distinguish NS with the rest of the users, using ROC curve analysis. This study will pave the way for the utilizing the potential of exosomes/miRNAs as a novel theranostic agents in understanding the lung injury caused by tobacco smoking and vaping.

scholarly journals Exosomal microRNAs are novel circulating biomarkers in cigarette, waterpipe smokers, E-cigarette users and dual smokers

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Kameshwar P. Singh ◽  
Krishna P. Maremanda ◽  
Dongmei Li ◽  
Irfan Rahman
Keyword(s):  
Differential Expression ◽  
Tobacco Smoking ◽  
Dna Analysis ◽  
Electronic Cigarettes ◽  
Differential Expression Analysis ◽  
High Sensitivity ◽  
Gene Set Enrichment Analysis ◽  
Differentially Expressed ◽  
Cigarette Smokers ◽  
Non Coding Rnas

Abstract Background Electronic cigarettes (e-cigs) vaping, cigarette smoke, and waterpipe tobacco smoking are associated with various cardiopulmonary diseases. microRNAs are present in higher concentration in exosomes that play an important role in various physiological and pathological functions. We hypothesized that the non-coding RNAs transcript may serve as susceptibility to disease biomarkers by smoking and vaping. Methods Plasma exosomes/EVs from cigarette smokers, waterpipe smokers and dual smokers (cigarette and waterpipe) were characterized for their size, morphology and TEM, Nanosight and immunoblot analysis. Exosomal RNA was used for small RNA library preparation and the library was quantified using the High Sensitivity DNA Analysis on the Agilent 2100 Bioanalyzer system and sequenced using the Illumina NextSeq 500 and were converted to fastq format for mapping genes. Results Enrichment of various non-coding RNAs that include microRNAs, tRNAs, piRNAs, snoRNAs, snRNAs, Mt-tRNAs, and other biotypes are shown in exosomes. A comprehensive differential expression analysis of miRNAs, tRNAs and piRNAs showed significant changes across different pairwise comparisons. The seven microRNAs that were common and differentially expressed of when all the smoking and vaping groups were compared with non-smokers (NS) are hsa-let-7a-5p, hsa-miR-21-5p, hsa-miR-29b-3p, hsa-let-7f-5p, hsa-miR-143-3p, hsa-miR-30a-5p and hsa-let-7i-5p. The e-cig vs. NS group has differentially expressed 5 microRNAs (hsa-miR-224-5p, hsa-miR-193b-3p, hsa-miR-30e-5p, hsa-miR-423-3p, hsa-miR-365a-3p, and hsa-miR-365b-3p), which are not expressed in other three groups. Gene set enrichment analysis of microRNAs showed significant changes in the top six enriched functions that consisted of biological pathway, biological process, molecular function, cellular component, site of expression and transcription factor in all the groups. Further, the pairwise comparison of tRNAs and piRNA in all these groups revealed significant changes in their expressions. Conclusions Plasma exosomes of cigarette smokers, waterpipe smokers, e-cig users and dual smokers have common differential expression of microRNAs which may serve to distinguish smoking and vaping subjects from NS. Among them has-let-7a-5p has high sensitivity and specificity to distinguish NS with the rest of the users, using ROC curve analysis. These findings will pave the way for the utilizing the potential of exosomes/miRNAs as a novel theranostic agents in lung injury and disease caused by tobacco smoking and vaping.

scholarly journals Construction of a ceRNA network in glioma and analysis of its clinical significance

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Guangdong Liu ◽  
Haihong Li ◽  
Wenyang Ji ◽  
Haidong Gong ◽  
Yan Jiang ◽  
...  
Keyword(s):  
Differential Expression ◽  
Expression Analysis ◽  
Molecular Mechanisms ◽  
Cox Regression ◽  
Differential Expression Analysis ◽  
Gene Set Enrichment Analysis ◽  
Normal Brain ◽  
Central Nervous System Tumor ◽  
Cox Regression Analysis ◽  
Cerna Network

Abstract Background Glioma is the most common central nervous system tumor with a poor survival rate and prognosis. Previous studies have found that long non-coding RNA (lncRNA) and competitive endogenous RNA (ceRNA) play important roles in regulating various tumor mechanisms. We obtained RNA-Seq data of glioma and normal brain tissue samples from TCGA and GTEx databases and extracted the lncRNA and mRNA expression data. Further, we analyzed these data using weighted gene co-expression network analysis and differential expression analysis, respectively. Differential expression analysis was also carried out on the mRNA data from the GEO database. Further, we predicted the interactions between lncRNA, miRNA, and targeted mRNA. Using the CGGA data to perform univariate and multivariate Cox regression analysis on mRNA. Results We constructed a Cox proportional hazard regression model containing four mRNAs and performed immune infiltration analysis. Moreover, we also constructed a ceRNA network including 21 lncRNAs, two miRNAs, and four mRNAs, and identified seven lncRNAs related to survival that have not been previously studied in gliomas. Through the gene set enrichment analysis, we found four lncRNAs that may have a significant role in tumors and should be explored further in the context of gliomas. Conclusions In short, we identified four lncRNAs with research value for gliomas, constructed a ceRNA network in gliomas, and developed a prognostic prediction model. Our research enhances our understanding of the molecular mechanisms underlying gliomas, providing new insights for developing targeted therapies and efficiently evaluating the prognosis of gliomas.

Cross-species Transcriptomes Reveal Species-specific and Shared Molecular Adaptations for Plants Development on Iron-rich Rocky Outcrops Soils

2021 ◽  
Author(s):  
Mariana Costa Dias ◽  
Cecílio Caldeira ◽  
Markus Gastauer ◽  
Silvio Ramos ◽  
Guilherme Oliveira
Keyword(s):  
Circadian Rhythm ◽  
Differential Expression ◽  
Native Species ◽  
Molecular Mechanisms ◽  
Light Stimulus ◽  
Differential Expression Analysis ◽  
Common Mechanism ◽  
Dependent Manner ◽  
Rocky Outcrops ◽  
Species Specific

Abstract BackgroundCanga is the Brazilian term for the savanna-like vegetation harboring several endemic species on iron-rich rocky outcrops, usually considered for mining activities. Parkia platycephala Benth. and Stryphnodendron pulcherrimum (Willd.) Hochr. naturally occur in the cangas of Serra dos Carajás (eastern Amazonia, Brazil) and the surrounding forest, indicating high phenotypic plasticity. The morphological and physiological mechanisms of the plants’ establishment in the canga environment are well studied, but the molecular adaptative responses are still unknown. We aimed to identify molecular mechanisms that allow the establishment of these plants in the canga environment.ResultsPlants were grown in canga and forest substrates collected in the Carajás Mineral Province. RNA was extracted from pooled leaf tissue, and RNA-seq paired-end reads were assembled into representative transcriptomes for P. platycephala and S. pulcherrimum containing 31,728 and 31,311 primary transcripts, respectively. We identified both species-specific and core molecular responses in plants grown in the canga substrate using differential expression analyses. In the species-specific analysis, we identified 1,112 and 838 differentially expressed genes for P. platycephala and S. pulcherrimum, respectively. Enrichment analyses showed unique biological processes and metabolic pathways affected for each species. Comparative differential expression analysis was based on shared single-copy orthologs. The overall pattern of ortholog expression was species-specific. Even so, almost 300 altered genes were identified between plants in canga and forest substrates, responding the same way in both species. The genes were functionally associated with the response to light stimulus and the circadian rhythm pathway.ConclusionsPlants possess species-specific adaptative responses to cope with the substrates. Our results also suggest that plants adapted to both canga and forest environments can adjust the circadian rhythm in a substrate-dependent manner. The circadian clock gene modulation might be a central mechanism regulating the plants’ development in the canga substrate in the studied legume species. The mechanism may be shared as a common mechanism to abiotic stress compensation in other native species.

Abstract 545: Differential mRNA Expression is Influenced by Apolipoprotein A-I in Order to Promote Foam Cell Regression

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Elisa C Maruko ◽  
Hao Xu ◽  
Sushma Kaul ◽  
Brian J Capaldo ◽  
Nathalie Pamir ◽  
...  
Keyword(s):  
Gene Expression ◽  
Differential Expression ◽  
Expression Analysis ◽  
Immune Cell ◽  
Foam Cell ◽  
Differential Expression Analysis ◽  
Ldl Receptor ◽  
Gene Set Enrichment Analysis ◽  
Control Group ◽  
Apolipoprotein A

Atherosclerosis is a disease of both lipids and inflammatory immune cells. More specifically, elevated plasma levels of low-density lipoproteins (LDL) leads to migration of circulating monocytes into the artery wall. Lipid loaded monocyte cells subsequently proliferate in the arterial walls becoming macrophage foam cells; a hallmark of atherosclerotic lesions. A proposed mechanism of the protective effects of high-density lipoprotein (HDL) is apolipoprotein A-I (apo A-I) acting as a mediator of cholesterol efflux and subsequent foam cell regression. To better understand the biological changes stimulated by apo A-I treatment, differential expression analysis of microarray data was performed on spleen cells from apo A-I treated mice. LDL receptor null (LDLr -/- ) and LDL receptor and apo A-I null (LDLr -/- , apoA-I -/- ) mice were fed a western diet consisting of 0.2% cholesterol and 42% of calories as fat for 12 weeks. After 6 weeks of diet, a subset of mice for each genotype was subcutaneously injected with 200 micrograms of apo A-I 3 times a week for the remaining 6 weeks. The control group mice were subcutaneously injected with 200 micrograms of saline or BSA. Spleen cell RNA was isolated, purified, and analyzed for differential expression analysis using Illumina BeadArray Microarray Technology Analysis. Individual gene expression analysis for LDLr -/- , apoA-I -/- apo A-I treated mice showed 281 significantly differentially expressed genes compared to BSA treated mice. LDLr -/- A-I treated mice had 1502. Of the significant genes, 189 intersected across both genotypes. LDLr -/- , apoA-I -/- A-I mice showed 73 up-regulated and 116 down-regulated genes. Similarly, LDLr -/- A-I mice had 71 up-regulated and 118 down-regulated. One-directional Gene Set Enrichment Analysis (GSEA) of LDLr -/- , apoA-I -/- A-I mice revealed 49 significant pathways while a total of 63 were found for LDLr -/- . Of these pathways, 21 were up-regulated and 13 were down-regulated in both genotypes. Eight of the top 10 most significant up-regulated pathways in both genotypes were immune cell related. Their functions involve receptor, adhesion, and chemokine signaling. Overall, preliminary analysis suggests A-I treatment induces similar gene expression changes across different genotypes.

scholarly journals Easy and efficient ensemble gene set testing with EGSEA

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 2010 ◽  
Author(s):  
Monther Alhamdoosh ◽  
Charity W. Law ◽  
Luyi Tian ◽  
Julie M. Sheridan ◽  
Milica Ng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Differential Expression ◽  
Expression Analysis ◽  
Differential Expression Analysis ◽  
Enrichment Analysis ◽  
Gene Set Enrichment Analysis ◽  
Gene Set ◽  
P Gene ◽  
Wide Range ◽  
Gene Set Testing

Gene set enrichment analysis is a popular approach for prioritising the biological processes perturbed in genomic datasets. The Bioconductor project hosts over 80 software packages capable of gene set analysis. Most of these packages search for enriched signatures amongst differentially regulated genes to reveal higher level biological themes that may be missed when focusing only on evidence from individual genes. With so many different methods on offer, choosing the best algorithm and visualization approach can be challenging. The EGSEA package solves this problem by combining results from up to 12 prominent gene set testing algorithms to obtain a consensus ranking of biologically relevant results.This workflow demonstrates how EGSEA can extend limma-based differential expression analyses for RNA-seq and microarray data using experiments that profile 3 distinct cell populations important for studying the origins of breast cancer. Following data normalization and set-up of an appropriate linear model for differential expression analysis, EGSEA builds gene signature specific indexes that link a wide range of mouse or human gene set collections obtained from MSigDB, GeneSetDB and KEGG to the gene expression data being investigated. EGSEA is then configured and the ensemble enrichment analysis run, returning an object that can be queried using several S4 methods for ranking gene sets and visualizing results via heatmaps, KEGG pathway views, GO graphs, scatter plots and bar plots. Finally, an HTML report that combines these displays can fast-track the sharing of results with collaborators, and thus expedite downstream biological validation. EGSEA is simple to use and can be easily integrated with existing gene expression analysis pipelines for both human and mouse data.

scholarly journals RNA sequencing of Stentor cell fragments reveals key processes underlying cellular regeneration

2017 ◽  
Author(s):  
Henning Onsbring Gustafson ◽  
Mahwash Jamy ◽  
Thijs J. G. Ettema
Keyword(s):  
Differential Expression ◽  
Rna Sequencing ◽  
Molecular Mechanisms ◽  
Anterior Part ◽  
Differential Expression Analysis ◽  
Biological Studies ◽  
Ability To Regenerate ◽  
Cellular Regeneration ◽  
A Cell ◽  
Cell Fragments

SummaryWhile ciliates of the genus Stentor are known for their ability to regenerate when their cells are damaged or even fragmented, the physical and molecular mechanisms underlying this process are poorly understood. To identify genes involved in the regenerative capability of Stentor cells, RNA sequencing of individual Stentor polymorphus cell fragments was performed. After splitting a cell over the anterior-posterior axis, the posterior fragment has to regenerate the oral apparatus, while the anterior part needs to regenerate the hold fast. Altogether, differential expression analysis of both posterior and anterior S. polymorphus cell fragments for four different post-split time points revealed over 10,000 up-regulated genes throughout the regeneration process. Among these, genes involved in cell signaling, microtubule-based movement and cell cycle regulation seemed to be particularly important during cellular regeneration. We identified roughly nine times as many up-regulated genes in regenerating S. polymorphus posterior fragments as compared to anterior fragments, indicating that regeneration of the anterior oral apparatus is a complex process that involves many genes. Our analyses identified several expanded groups of genes such as dual-specific tyrosine-(Y)-phosphorylation regulated kinases and MORN domain containing proteins that seemingly act as key-regulators of cellular regeneration. In agreement with earlier morphological and cell biological studies, our differential expression analyses indicate that cellular regeneration and vegetative division share many similarities.

scholarly journals Impact of sickle cell trait hemoglobin on the intraerythrocytic transcriptional program of Plasmodium falciparum

2021 ◽  
Author(s):  
Joseph W Saelens ◽  
Jens E.V. Petersen ◽  
Elizabeth Freedman ◽  
Robert C Moseley ◽  
Drissa Konate ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Differential Expression ◽  
Molecular Mechanisms ◽  
Sickle Cell Trait ◽  
Differential Expression Analysis ◽  
Protein Export ◽  
In Vitro Cultivation ◽  
Transcriptional Program ◽  
Transcriptional Responses

Sickle-trait hemoglobin (HbAS) confers near-complete protection from severe, life-threatening falciparum malaria in African children. Despite this clear protection, the molecular mechanisms by which HbAS confers these protective phenotypes remain incompletely understood. As a forward genetic screen for aberrant parasite transcriptional responses associated with parasite neutralization in HbAS red blood cells (RBCs), we performed comparative transcriptomic analyses of Plasmodium falciparum in normal (HbAA) and HbAS erythrocytes during both in vitro cultivation of reference parasite strains and naturally-occurring P. falciparum infections in Malian children with HbAA or HbAS. During in vitro cultivation, parasites matured normally in HbAS RBCs, and the temporal expression was largely unperturbed of the highly ordered transcriptional program that underlies the parasites maturation throughout the intraerythrocytic development cycle (IDC). However, differential expression analysis identified hundreds of transcripts aberrantly expressed in HbAS, largely occurring late in the IDC. Surprisingly, transcripts encoding members of the Maurers clefts were overexpressed in HbAS despite impaired parasite protein export in these RBCs, while parasites in HbAS RBCs underexpressed transcripts associated with the endoplasmic reticulum and those encoding serine repeat antigen proteases that promote parasite egress. Analyses of P. falciparum transcriptomes from 32 children with uncomplicated malaria identified stage-specific differential expression: among infections composed of ring-stage parasites, only cyclophilin 19B was underexpressed in children with HbAS, while trophozoite-stage infections identified a range of differentially-expressed transcripts, including downregulation in HbAS of several transcripts associated with severe malaria in collateral studies. Collectively, our comparative transcriptomic screen in vitro and in vivo indicates that P. falciparum adapts to HbAS by altering its protein chaperone and folding machinery, oxidative stress response, and protein export machinery. Because HbAS consistently protects from severe P. falciparum, modulation of these responses may offer avenues by which to neutralize P. falciparum parasites.

scholarly journals Sex differences in the human brain transcriptome of cases with schizophrenia

2020 ◽  
Author(s):  
Gabriel E. Hoffman ◽  
Yixuan Ma ◽  
Kelsey S. Montgomery ◽  
Jaroslav Bendl ◽  
Manoj Kumar Jaiswal ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sex Differences ◽  
Differential Expression ◽  
Large Scale ◽  
Molecular Mechanisms ◽  
Age Of Onset ◽  
Differential Expression Analysis ◽  
Gene Expression Signature ◽  
Synaptic Organization ◽  
Expression Signature

AbstractWhile schizophrenia differs between males and females in age of onset, symptomatology and the course of the disease, the molecular mechanisms underlying these differences remain uncharacterized. In order to address questions about the sex-specific effects of schizophrenia, we performed a large-scale transcriptome analysis of RNA-seq data from 437 controls and 341 cases from two distinct cohorts from the CommonMind Consortium. Analysis across the cohorts identifies a reproducible gene expression signature of schizophrenia that is highly concordant with previous work. Differential expression across sex is reproducible across cohorts and identifies X- and Y-linked genes, as well as those involved in dosage compensation. Intriguingly, the sex expression signature is also enriched for genes involved in neurexin family protein binding and synaptic organization. Differential expression analysis testing a sex-by-diagnosis interaction effect did not identify any genome-wide signature after multiple testing corrections. Gene coexpression network analysis was performed to reduce dimensionality and elucidate interactions among genes. We found enrichment of co-expression modules for sex-by-diagnosis differential expression signatures, which were highly reproducible across the two cohorts and involve a number of diverse pathways, including neural nucleus development, neuron projection morphogenesis, and regulation of neural precursor cell proliferation. Overall, our results indicate that the effect size of sex differences in schizophrenia gene expression signatures is small and underscore the challenge of identifying robust sex-by-diagnosis signatures, which will require future analyses in larger cohorts.

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