Erythrocyte microRNA sequencing reveals differential expression in relapsing-remitting multiple sclerosis

Kira Groen, Vicki E. Maltby, Rodney A. Lea, Katherine A. Sanders, J. Lynn Fink, Rodney J. Scott, Lotti Tajouri, Jeannette Lechner-Scott

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Abstract

Background: There is a paucity of knowledge concerning erythrocytes in the aetiology of Multiple Sclerosis (MS) despite their potential to contribute to disease through impaired antioxidant capacity and altered haemorheological features. Several studies have identified an abundance of erythrocyte miRNAs and variable profiles associated with disease states, such as sickle cell disease and malaria. The aim of this study was to compare the erythrocyte miRNA profile of relapsing-remitting MS (RRMS) patients to healthy sex- and age-matched controls. Methods: Erythrocytes were purified by density-gradient centrifugation and RNA was extracted. Following library preparation, samples were run on a HiSeq4000 Illumina instrument (paired-end 100 bp sequencing). Sequenced erythrocyte miRNA profiles (9 patients and 9 controls) were analysed by DESeq2. Differentially expressed miRNAs were validated by RT-qPCR using miR-152-3p as an endogenous control and replicated in a larger cohort (20 patients and 18 controls). After logarithmic transformation, differential expression was determined by two-tailed unpaired t-tests. Logistic regression analysis was carried out and receiver operating characteristic (ROC) curves were generated to determine biomarker potential. Results: A total of 236 erythrocyte miRNAs were identified. Of twelve differentially expressed miRNAs in RRMS two showed increased expression (adj. p < 0.05). Only modest fold-changes were evident across differentially expressed miRNAs. RT-qPCR confirmed differential expression of miR-30b-5p (0.61 fold, p < 0.05) and miR-3200-3p (0.36 fold, p < 0.01) in RRMS compared to healthy controls. Relative expression of miR-3200-5p (0.66 fold, NS p = 0.096) also approached significance. MiR-3200-5p was positively correlated with cognition measured by audio-recorded cognitive screen (r = 0.60; p < 0.01). MiR-3200-3p showed greatest biomarker potential as a single miRNA (accuracy = 75.5%, p < 0.01, sensitivity = 72.7%, specificity = 84.0%). Combining miR-3200-3p, miR-3200-5p, and miR-30b-5p into a composite biomarker increased accuracy to 83.0% (p < 0.05), sensitivity to 77.3%, and specificity to 88.0%. Conclusions: This is the first study to report differences in erythrocyte miRNAs in RRMS. While the role of miRNAs in erythrocytes remains to be elucidated, differential expression of erythrocyte miRNAs may be exploited as biomarkers and their potential contribution to MS pathology and cognition should be further investigated.

Original languageEnglish
Article number48
Number of pages12
JournalBMC Medical Genomics
Volume11
Issue number1
DOIs
Publication statusPublished - 21 May 2018

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Relapsing-Remitting Multiple Sclerosis
MicroRNAs
Erythrocytes
Biomarkers
Cognition
Multiple Sclerosis
Density Gradient Centrifugation
Sickle Cell Anemia
ROC Curve
Malaria
Libraries
Antioxidants
Logistic Models
Regression Analysis

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Groen, K., Maltby, V. E., Lea, R. A., Sanders, K. A., Fink, J. L., Scott, R. J., ... Lechner-Scott, J. (2018). Erythrocyte microRNA sequencing reveals differential expression in relapsing-remitting multiple sclerosis. BMC Medical Genomics, 11(1), [48]. https://doi.org/10.1186/s12920-018-0365-7
Groen, Kira ; Maltby, Vicki E. ; Lea, Rodney A. ; Sanders, Katherine A. ; Fink, J. Lynn ; Scott, Rodney J. ; Tajouri, Lotti ; Lechner-Scott, Jeannette. / Erythrocyte microRNA sequencing reveals differential expression in relapsing-remitting multiple sclerosis. In: BMC Medical Genomics. 2018 ; Vol. 11, No. 1.
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title = "Erythrocyte microRNA sequencing reveals differential expression in relapsing-remitting multiple sclerosis",
abstract = "Background: There is a paucity of knowledge concerning erythrocytes in the aetiology of Multiple Sclerosis (MS) despite their potential to contribute to disease through impaired antioxidant capacity and altered haemorheological features. Several studies have identified an abundance of erythrocyte miRNAs and variable profiles associated with disease states, such as sickle cell disease and malaria. The aim of this study was to compare the erythrocyte miRNA profile of relapsing-remitting MS (RRMS) patients to healthy sex- and age-matched controls. Methods: Erythrocytes were purified by density-gradient centrifugation and RNA was extracted. Following library preparation, samples were run on a HiSeq4000 Illumina instrument (paired-end 100 bp sequencing). Sequenced erythrocyte miRNA profiles (9 patients and 9 controls) were analysed by DESeq2. Differentially expressed miRNAs were validated by RT-qPCR using miR-152-3p as an endogenous control and replicated in a larger cohort (20 patients and 18 controls). After logarithmic transformation, differential expression was determined by two-tailed unpaired t-tests. Logistic regression analysis was carried out and receiver operating characteristic (ROC) curves were generated to determine biomarker potential. Results: A total of 236 erythrocyte miRNAs were identified. Of twelve differentially expressed miRNAs in RRMS two showed increased expression (adj. p < 0.05). Only modest fold-changes were evident across differentially expressed miRNAs. RT-qPCR confirmed differential expression of miR-30b-5p (0.61 fold, p < 0.05) and miR-3200-3p (0.36 fold, p < 0.01) in RRMS compared to healthy controls. Relative expression of miR-3200-5p (0.66 fold, NS p = 0.096) also approached significance. MiR-3200-5p was positively correlated with cognition measured by audio-recorded cognitive screen (r = 0.60; p < 0.01). MiR-3200-3p showed greatest biomarker potential as a single miRNA (accuracy = 75.5{\%}, p < 0.01, sensitivity = 72.7{\%}, specificity = 84.0{\%}). Combining miR-3200-3p, miR-3200-5p, and miR-30b-5p into a composite biomarker increased accuracy to 83.0{\%} (p < 0.05), sensitivity to 77.3{\%}, and specificity to 88.0{\%}. Conclusions: This is the first study to report differences in erythrocyte miRNAs in RRMS. While the role of miRNAs in erythrocytes remains to be elucidated, differential expression of erythrocyte miRNAs may be exploited as biomarkers and their potential contribution to MS pathology and cognition should be further investigated.",
author = "Kira Groen and Maltby, {Vicki E.} and Lea, {Rodney A.} and Sanders, {Katherine A.} and Fink, {J. Lynn} and Scott, {Rodney J.} and Lotti Tajouri and Jeannette Lechner-Scott",
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Erythrocyte microRNA sequencing reveals differential expression in relapsing-remitting multiple sclerosis. / Groen, Kira; Maltby, Vicki E.; Lea, Rodney A.; Sanders, Katherine A.; Fink, J. Lynn; Scott, Rodney J.; Tajouri, Lotti; Lechner-Scott, Jeannette.

In: BMC Medical Genomics, Vol. 11, No. 1, 48, 21.05.2018.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Erythrocyte microRNA sequencing reveals differential expression in relapsing-remitting multiple sclerosis

AU - Groen, Kira

AU - Maltby, Vicki E.

AU - Lea, Rodney A.

AU - Sanders, Katherine A.

AU - Fink, J. Lynn

AU - Scott, Rodney J.

AU - Tajouri, Lotti

AU - Lechner-Scott, Jeannette

PY - 2018/5/21

Y1 - 2018/5/21

N2 - Background: There is a paucity of knowledge concerning erythrocytes in the aetiology of Multiple Sclerosis (MS) despite their potential to contribute to disease through impaired antioxidant capacity and altered haemorheological features. Several studies have identified an abundance of erythrocyte miRNAs and variable profiles associated with disease states, such as sickle cell disease and malaria. The aim of this study was to compare the erythrocyte miRNA profile of relapsing-remitting MS (RRMS) patients to healthy sex- and age-matched controls. Methods: Erythrocytes were purified by density-gradient centrifugation and RNA was extracted. Following library preparation, samples were run on a HiSeq4000 Illumina instrument (paired-end 100 bp sequencing). Sequenced erythrocyte miRNA profiles (9 patients and 9 controls) were analysed by DESeq2. Differentially expressed miRNAs were validated by RT-qPCR using miR-152-3p as an endogenous control and replicated in a larger cohort (20 patients and 18 controls). After logarithmic transformation, differential expression was determined by two-tailed unpaired t-tests. Logistic regression analysis was carried out and receiver operating characteristic (ROC) curves were generated to determine biomarker potential. Results: A total of 236 erythrocyte miRNAs were identified. Of twelve differentially expressed miRNAs in RRMS two showed increased expression (adj. p < 0.05). Only modest fold-changes were evident across differentially expressed miRNAs. RT-qPCR confirmed differential expression of miR-30b-5p (0.61 fold, p < 0.05) and miR-3200-3p (0.36 fold, p < 0.01) in RRMS compared to healthy controls. Relative expression of miR-3200-5p (0.66 fold, NS p = 0.096) also approached significance. MiR-3200-5p was positively correlated with cognition measured by audio-recorded cognitive screen (r = 0.60; p < 0.01). MiR-3200-3p showed greatest biomarker potential as a single miRNA (accuracy = 75.5%, p < 0.01, sensitivity = 72.7%, specificity = 84.0%). Combining miR-3200-3p, miR-3200-5p, and miR-30b-5p into a composite biomarker increased accuracy to 83.0% (p < 0.05), sensitivity to 77.3%, and specificity to 88.0%. Conclusions: This is the first study to report differences in erythrocyte miRNAs in RRMS. While the role of miRNAs in erythrocytes remains to be elucidated, differential expression of erythrocyte miRNAs may be exploited as biomarkers and their potential contribution to MS pathology and cognition should be further investigated.

AB - Background: There is a paucity of knowledge concerning erythrocytes in the aetiology of Multiple Sclerosis (MS) despite their potential to contribute to disease through impaired antioxidant capacity and altered haemorheological features. Several studies have identified an abundance of erythrocyte miRNAs and variable profiles associated with disease states, such as sickle cell disease and malaria. The aim of this study was to compare the erythrocyte miRNA profile of relapsing-remitting MS (RRMS) patients to healthy sex- and age-matched controls. Methods: Erythrocytes were purified by density-gradient centrifugation and RNA was extracted. Following library preparation, samples were run on a HiSeq4000 Illumina instrument (paired-end 100 bp sequencing). Sequenced erythrocyte miRNA profiles (9 patients and 9 controls) were analysed by DESeq2. Differentially expressed miRNAs were validated by RT-qPCR using miR-152-3p as an endogenous control and replicated in a larger cohort (20 patients and 18 controls). After logarithmic transformation, differential expression was determined by two-tailed unpaired t-tests. Logistic regression analysis was carried out and receiver operating characteristic (ROC) curves were generated to determine biomarker potential. Results: A total of 236 erythrocyte miRNAs were identified. Of twelve differentially expressed miRNAs in RRMS two showed increased expression (adj. p < 0.05). Only modest fold-changes were evident across differentially expressed miRNAs. RT-qPCR confirmed differential expression of miR-30b-5p (0.61 fold, p < 0.05) and miR-3200-3p (0.36 fold, p < 0.01) in RRMS compared to healthy controls. Relative expression of miR-3200-5p (0.66 fold, NS p = 0.096) also approached significance. MiR-3200-5p was positively correlated with cognition measured by audio-recorded cognitive screen (r = 0.60; p < 0.01). MiR-3200-3p showed greatest biomarker potential as a single miRNA (accuracy = 75.5%, p < 0.01, sensitivity = 72.7%, specificity = 84.0%). Combining miR-3200-3p, miR-3200-5p, and miR-30b-5p into a composite biomarker increased accuracy to 83.0% (p < 0.05), sensitivity to 77.3%, and specificity to 88.0%. Conclusions: This is the first study to report differences in erythrocyte miRNAs in RRMS. While the role of miRNAs in erythrocytes remains to be elucidated, differential expression of erythrocyte miRNAs may be exploited as biomarkers and their potential contribution to MS pathology and cognition should be further investigated.

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