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Identification of biomarkers for amyotrophic lateral sclerosis by comprehensive analysis of exosomal mRNAs in human cerebrospinal fluid.

ABSTRACT: BACKGROUND:Exosomes are a subset of extracellular vesicles 30-200?nm in diameter secreted from cells, which contain functional mRNAs and microRNAs. Cerebrospinal fluid (CSF) is the primary source for liquid biopsy to examine diseases in central nervous system. To date, there is no available method to analyze exosomal mRNAs comprehensively in human CSF. METHODS:The main purpose of this study is to established the methodology of comprehensive analysis of exosomal mRNAs in CSF by a highly sensitive next-generation sequencing. The signatures of CSF exosomal mRNAs were then compared between four normal healthy donors and four sporadic amyotrophic lateral sclerosis patients to identify disease-related biomarkers. Differentially expressed genes were identified by DESeq2. RESULTS:RNA sequencing from CSF exosomes was successfully performed, that was demonstrated by the high pearson's product-moment correlation coefficient (r?=?0.993) in the technical replicates. Also, position coverage analysis revealed that most detected mRNAs retained their integrity throughout their full-length in CSF exosomes. In CSF exosomes from normal healthy donors, an average of 14,807 genes were detected, of which 4580 genes were commonly detected among four individuals, including neuron-enriched genes such as TUBB3 and CAMK2A. In comparison with exosomal mRNAs in CSF from four patients with amyotrophic lateral sclerosis, 543 genes were significantly changed, as represented by CUEDC2. Gene Ontology analysis and pathway analysis with these genes revealed functional enrichment of ubiquitin-proteasome pathway, oxidative stress response, and unfolded protein response. These pathways are related to pathomechanisms of amyotrophic lateral sclerosis. CONCLUSION:We successfully established the methodology of comprehensive analysis of exosomal mRNAs in human CSF. It was shown to be useful to identify disease biomarkers for central nervous system. Several genes, such as CUEDC2, in CSF exosomes were suggested to be candidate disease biomarkers for amyotrophic lateral sclerosis.

SUBMITTER: Otake K

PROVIDER: S-EPMC6329125 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Identification of biomarkers for amyotrophic lateral sclerosis by comprehensive analysis of exosomal mRNAs in human cerebrospinal fluid.

Otake Kentaro K Kamiguchi Hidenori H Hirozane Yoshihiko Y

BMC medical genomics 20190110 1

<h4>Background</h4>Exosomes are a subset of extracellular vesicles 30-200 nm in diameter secreted from cells, which contain functional mRNAs and microRNAs. Cerebrospinal fluid (CSF) is the primary source for liquid biopsy to examine diseases in central nervous system. To date, there is no available method to analyze exosomal mRNAs comprehensively in human CSF.<h4>Methods</h4>The main purpose of this study is to established the methodology of comprehensive analysis of exosomal mRNAs in CSF by a h ...[more]

PMID: 30630471

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Project description:The present study used comparative proteomic analysis of cerebrospinal fluid (CSF) in amyotrophic lateral sclerosis (ALS) patients in order to identify proteins that may act as diagnostic biomarkers and indicators of the pathogenesis of ALS. This analysis was performed using isobaric tags for relative and absolute quantitation (iTRAQ) technology, coupled with 2-dimensional liquid chromatography/mass spectrometry. Database for Annotation, Visualization and Integrated Discovery software was utilized for bioinformatic analysis of the data. Following this, western blotting was performed in order to examine the expression of 3 candidate proteins in ALS patients compared with healthy individuals [as a normal control (NC) group] or patients with other neurological disease (OND); these proteins were insulin-like growth factor II (IGF-2), glutamate receptor 4 (GRIA4) and leucine-rich α-2-glycoprotein 1 (LRG1). Clinical data, including gender, age, disease duration and ALS functional rating scale (ALSFRS-R) score, were also collected in the ALS patients. Multiple linear regression analysis was performed between the clinical data and the results of western blot analysis. A total of 248 distinct proteins were identified in the ALS and NC groups, amongst which a significant difference could be identified in 35 proteins; of these, 21 proteins were downregulated and 14 were upregulated. These differentially-expressed proteins were thus revealed to be associated with ALS. The western blot analysis confirmed a proportion of the data attained in the iTRAQ analysis, revealing the differential protein expression of IGF-2 and GRIA4 between the ALS and NC groups. IGF-2 was significantly downregulated in ALS patients (P=0.017) and GRIA4 was significantly upregulated (P=0.016). These results were subsequently validated in the 35-patient ALS and OND groups (P=0.002), but no significant difference was identified in LRG1 expression between these groups. GRIA4 protein expression was higher in male than female patients and was positively correlated with the ALSFRS-R score, meaning that GRIA4 expression was negatively correlated with the severity of ALS, while IGF-2 and LRG1 expression did not correlate with any clinical data. The present study thus demonstrated that GRIA4 expression levels, as a marker of severity, may be used as a reference for the timing of treatment, and that IGF-2 may serve as an effective biomarker of ALS progression.

Dataset Information

Identification of biomarkers for amyotrophic lateral sclerosis by comprehensive analysis of exosomal mRNAs in human cerebrospinal fluid.

Publications

Identification of biomarkers for amyotrophic lateral sclerosis by comprehensive analysis of exosomal mRNAs in human cerebrospinal fluid.

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