title: Next-generation sequencing identifies altered whole blood microRNAs in neuromyelitis optica spectrum disorder which may permit discrimination from multiple sclerosis.
abstract: BACKGROUND
  Neuromyelitis optica spectrum disorder (NMOSD) and multiple sclerosis (MS) have a similar clinical phenotype but represent distinct diseases, requiring different therapies. MicroRNAs (miRNAs) are short non-coding RNAs whose expression profiles can serve as diagnostic biomarkers and which may be involved in the pathophysiology of neuroinflammatory diseases. Here, we analyzed miRNA profiles in serum and whole blood of patients with NMOSD and clinically isolated syndrome (CIS)/relapsing-remitting MS (RRMS) as well as healthy controls by next-generation sequencing (NGS).
  
  
  METHODS
  MiRNA expression profiles were determined by NGS in sera of patients with aquaporin-4 antibody-positive NMOSD (n = 20), CIS/RRMS (n = 20), and healthy controls (n = 20) and in whole blood of patients with NMOSD (n = 11), CIS/RRMS (n = 60), and healthy controls (n = 43). Differentially expressed miRNAs were calculated by analysis of variance and t tests. All significance values were corrected for multiple testing. Selected miRNAs were validated in whole blood of patients with NMOSD (n = 18) and CIS/RRMS (n = 19) by quantitative real-time polymerase chain reaction (qRT-PCR).
  
  
  RESULTS
  None of 261 miRNAs detected in serum but 178 of 416 miRNAs detected in whole blood showed significantly different expression levels among the three groups. Pairwise comparisons revealed 115 (NMOSD vs. CIS/RRMS), 141 (NMOSD vs. healthy controls), and 44 (CIS/RRMS vs. healthy controls) miRNAs in whole blood with significantly different expression levels. qRT-PCR confirmed different expression levels in whole blood of patients with NMOSD and CIS/RRMS for 9 out of 10 exemplarily chosen miRNAs. In silico enrichment analysis demonstrated an accumulation of altered miRNAs in NMOSD in particular in CD15(+) cells (i.e., neutrophils and eosinophils).
  
  
  CONCLUSIONS
  This study identifies a set of miRNAs in whole blood, which may have the potential to discriminate NMOSD from CIS/RRMS and healthy controls. In contrast, miRNA profiles in serum do not appear to be promising diagnostic biomarkers for NMOSD. Enrichment of altered miRNAs in CD15(+) neutrophils and eosinophils, which were previously implicated in the pathophysiology of NMOSD, suggests that miRNAs could be involved in the regulation of these cells in NMOSD.
authors.affiliation: Clinical Bioinformatics, Saarland University, Saarbrücken, Germany. andreas.keller@ccb.uni-saarland.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Keller
authors.affiliation: Human Genetics, Saarland University, Homburg, Germany. p.leidinger@mx.uni-saarland.de.
authors.forename: Petra
authors.initials: P
authors.lastname: Leidinger
authors.affiliation: Human Genetics, Saarland University, Homburg, Germany. hgemee@uniklinik-saarland.de.
authors.forename: Eckart
authors.initials: E
authors.lastname: Meese
authors.affiliation: Internal Medicine III, Heidelberg University Hospital, Heidelberg, Germany. jan.haas@med.uni-heidelberg.de.
authors.forename: Jan
authors.initials: J
authors.lastname: Haas
authors.affiliation: Clinical Bioinformatics, Saarland University, Saarbrücken, Germany. c.backes@mx.uni-saarland.de.
authors.forename: Christina
authors.initials: C
authors.lastname: Backes
authors.affiliation: Department of Neurology, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany. ludwig.rasche@charite.de.|NeuroCure Clinical Research Center, Charité-Universitätsmedizin Berlin, Berlin, Germany. ludwig.rasche@charite.de.|Clinical and Experimental Multiple Sclerosis Research Center, Charité-Universitätsmedizin Berlin, Berlin, Germany. ludwig.rasche@charite.de.
authors.forename: Ludwig
authors.initials: L
authors.lastname: Rasche
authors.affiliation: NeuroCure Clinical Research Center, Charité-Universitätsmedizin Berlin, Berlin, Germany. janina.behrens@charite.de.|Clinical and Experimental Multiple Sclerosis Research Center, Charité-Universitätsmedizin Berlin, Berlin, Germany. janina.behrens@charite.de.
authors.forename: Janina R
authors.initials: JR
authors.lastname: Behrens
authors.affiliation: Department of Neurology, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany. catherina.pfuhl@charite.de.|NeuroCure Clinical Research Center, Charité-Universitätsmedizin Berlin, Berlin, Germany. catherina.pfuhl@charite.de.|Clinical and Experimental Multiple Sclerosis Research Center, Charité-Universitätsmedizin Berlin, Berlin, Germany. catherina.pfuhl@charite.de.
authors.forename: Catherina
authors.initials: C
authors.lastname: Pfuhl
authors.affiliation: Department of Neurology, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany. katharina.wakonig@charite.de.|NeuroCure Clinical Research Center, Charité-Universitätsmedizin Berlin, Berlin, Germany. katharina.wakonig@charite.de.|Clinical and Experimental Multiple Sclerosis Research Center, Charité-Universitätsmedizin Berlin, Berlin, Germany. katharina.wakonig@charite.de.
authors.forename: Katharina
authors.initials: K
authors.lastname: Wakonig
authors.affiliation: Department of Neurology, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany. rene.giess@charite.de.|NeuroCure Clinical Research Center, Charité-Universitätsmedizin Berlin, Berlin, Germany. rene.giess@charite.de.|Clinical and Experimental Multiple Sclerosis Research Center, Charité-Universitätsmedizin Berlin, Berlin, Germany. rene.giess@charite.de.
authors.forename: René M
authors.initials: RM
authors.lastname: Gieß
authors.affiliation: Molecular Neuroimmunology Group, Department of Neurology, University of Heidelberg, Heidelberg, Germany. sjarius@gmx.net.
authors.forename: Sven
authors.initials: S
authors.lastname: Jarius
authors.affiliation: Internal Medicine III, Heidelberg University Hospital, Heidelberg, Germany. benjamin.meder@med.uni-heidelberg.de.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Meder
authors.affiliation: NeuroCure Clinical Research Center, Charité-Universitätsmedizin Berlin, Berlin, Germany. judith.bellmann-strobl@charite.de.|Clinical and Experimental Multiple Sclerosis Research Center, Charité-Universitätsmedizin Berlin, Berlin, Germany. judith.bellmann-strobl@charite.de.|Experimental and Clinical Research Center, Max Delbrueck Center for Molecular Medicine and Charité-Universitätsmedizin Berlin, Berlin, Germany. judith.bellmann-strobl@charite.de.
authors.forename: Judith
authors.initials: J
authors.lastname: Bellmann-Strobl
authors.affiliation: Department of Neurology, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany. friedemann.paul@charite.de.|NeuroCure Clinical Research Center, Charité-Universitätsmedizin Berlin, Berlin, Germany. friedemann.paul@charite.de.|Clinical and Experimental Multiple Sclerosis Research Center, Charité-Universitätsmedizin Berlin, Berlin, Germany. friedemann.paul@charite.de.|Experimental and Clinical Research Center, Max Delbrueck Center for Molecular Medicine and Charité-Universitätsmedizin Berlin, Berlin, Germany. friedemann.paul@charite.de.
authors.forename: Friedemann
authors.initials: F
authors.lastname: Paul
authors.affiliation: Department of Neurology, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany. florence.pache@charite.de.|NeuroCure Clinical Research Center, Charité-Universitätsmedizin Berlin, Berlin, Germany. florence.pache@charite.de.|Clinical and Experimental Multiple Sclerosis Research Center, Charité-Universitätsmedizin Berlin, Berlin, Germany. florence.pache@charite.de.
authors.forename: Florence C
authors.initials: FC
authors.lastname: Pache
authors.affiliation: Department of Neurology, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany. klemens.ruprecht@charite.de.|Clinical and Experimental Multiple Sclerosis Research Center, Charité-Universitätsmedizin Berlin, Berlin, Germany. klemens.ruprecht@charite.de.
authors.forename: Klemens
authors.initials: K
authors.lastname: Ruprecht
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D051401:Aquaporin 4 / Q000276:immunology; D019295:Computational Biology; D003198:Computer Simulation; D005260:Female; D015723:Gene Library; D006801:Humans; D016256:Lewis X Antigen / Q000378:metabolism; D008297:Male; D035683:MicroRNAs / Q000097:blood / Q000235:genetics*; D008875:Middle Aged; D009103:Multiple Sclerosis / Q000097:blood / Q000235:genetics*; D009471:Neuromyelitis Optica / Q000097:blood / Q000235:genetics*; D016133:Polymerase Chain Reaction; D017423:Sequence Analysis, RNA; D055815:Young Adult
chemical_list: D051401:Aquaporin 4; D016256:Lewis X Antigen; D035683:MicroRNAs