Background
Neuromyelitis optica (NMO) was formerly considered a variant of multiple sclerosis (MS) [
1,
2]. However, the discovery of a highly specific serum autoantibody against the aquaporin-4 (AQP4) water channel, which is present in 60–80 % of patients with NMO, clarified that NMO is a separate disease pathophysiologically distinct from MS [
3‐
5]. While the classical NMO phenotype consists of a longitudinally extensive transverse myelitis (LETM) and optic neuritis (ON), AQP4 antibody-positive patients can also present with isolated LETM, ON, or brainstem encephalitis. This spectrum of clinical manifestations is now referred to as NMO spectrum disorder (NMOSD) [
5,
6]. Because the clinical phenotype of NMOSD and MS may be similar, but each condition requires different therapeutic approaches, accurate differentiation of NMOSD and MS is highly relevant [
7]. Although magnetic resonance imaging and AQP4 antibody testing aid in the differential diagnosis, misdiagnosis of NMOSD with MS is not infrequent [
8,
9] and reliable biomarkers that distinguish between both entities would be desirable.
MicroRNAs (miRNAs) are single-stranded, short (20–24 nucleotide) regulatory RNAs that regulate gene expression at the posttranscriptional level. MiRNAs are present in stable form in the human blood, and blood-based miRNA expression profiles may hold promise as diagnostic biomarkers in various human diseases, including cancer and autoimmune diseases [
10]. Indeed, studies performed by others and our group indicate that miRNA expression profiles determined either in whole blood or purified blood cell subtypes could play a role as potential biomarkers for MS [
11‐
25]. Furthermore, miRNAs circulating in plasma were recently shown to be differentially expressed in different stages of MS (relapsing-remitting MS (RRMS) vs. secondary progressive MS) and were thus proposed as an easily accessible blood-based biomarker to monitor MS [
26].
MiRNA profiles have hitherto not been investigated in NMOSD and their role in the differentiation of NMOSD, and MS has not been explored so far. A possible role of miRNAs in the pathophysiology of NMOSD likewise remains elusive. We here report the results of a systematic comparative analysis of miRNA profiles in serum and whole blood of patients with NMOSD and clinically isolated syndrome (CIS)/RRMS as well as healthy controls using next-generation sequencing.
Discussion
Here, we report the results of a systematic comparative study of both miRNA profiles in serum and whole blood of patients with NMOSD, CIS/RRMS, and healthy controls. The key findings of this work are (i) an absence of any significantly different serum miRNAs after adjustment for multiple testing, (ii) the detection of a number of highly significantly deregulated miRNAs in whole blood in the three-group and the two-group comparisons of patients with NMOSD, CIS/RRMS, and healthy controls, (iii) the validation of selected whole blood miRNAs deregulated between patients with NMOSD and CIS/RRMS by qRT-PCR, and (iv) the identification of cell types, in particular, CD15+ neutrophils and eosinophils, and pathways in which deregulated whole blood miRNAs are enriched.
While miRNA expression profiles were previously often investigated in whole blood or leukocyte subsets, recent studies suggested that also non-cell-associated miRNAs circulating in blood and measured in serum or plasma could serve as biomarkers for cancer, inflammation, and neurological diseases, including MS [
26,
34‐
37]. Therefore, we initially set out to analyze miRNA expression profiles in serum of patients with NMOSD, CIS/RRMS, and healthy controls. Because the pathogenesis of AQP4 antibody-negative NMOSD spectrum disorders (NMOSD) may be different from that of AQP4 antibody-positive NMOSD [
8,
38], we aimed to reduce inhomogeneity within the group of patients with NMOSD and purposefully included only AQP4 antibody-positive patients in our study. The 261 miRNAs detected across all three groups in the serum NGS study confirm that miRNAs are present over a broad range of expression levels in human serum and define the landscape of human serum miRNAs. Likewise, the detectability of miRNAs in serum suggests that these miRNAs could in principle serve as disease biomarkers. Nevertheless, since none of the observed differences of the expression levels of serum miRNAs in the three-group and the two-group comparisons remained significant after correction for multiple testing, the potential of serum miRNA profiles as biomarkers for differentiation of NMOSD, CIS/RRMS, and healthy controls appears questionable.
Because more miRNAs were detectable in whole blood (n = 416) than in serum (n = 261), not all miRNAs expressed in blood cells appear to be released into serum. Alternatively, the concentration of some miRNAs in serum could also be below the detection limit of NGS. Interestingly, few miRNAs were only detected in serum but not in whole blood, compatible with an enhanced secretion of these miRNAs into serum.
MiRNA profiling by NGS in whole blood samples identified a set of differentially expressed miRNAs in the three-group comparison and in the two-group comparisons, which remained significant after correction for multiple testing. This suggests that whole blood is a more appropriate biospecimen for analyses of miRNAs as biomarkers for neuroinflammatory diseases than serum. Indeed, the high AUC values of the miRNAs, which separated best between patients with NMOSD and CIS/RRMS (hsa-miR-30b-5p; AUC 0.9) and patients with NMOSD and healthy controls (hsa-miR-15b-3p; AUC 0.98), indicate that the expression levels of these miRNAs in whole blood may potentially discriminate patients with NMOSD from patients with CIS/RRMS and healthy controls. Interestingly, hsa-miR-15b-3p was not only the most significantly different miRNA when comparing NMOSD and healthy controls but was also the miRNA that discriminated second best between NMOSD and CIS/RRMS. Downregulated expression of miR15b-3p in whole blood may thus be specifically associated with NMOSD. In both comparisons, NMOSD vs. healthy controls and NMOSD vs. CIS/RRMS, clearly more miRNAs were lower in NMOSD than in CIS/RRMS or healthy controls (Fig.
4a,
b) among the significantly deregulated miRNAs. Although the reasons for the downregulation of a number of miRNAs in NMOSD are unclear, this again differentiates NMOSD from CIS/RRMS, where most of the significantly deregulated miRNAs were higher in patients than healthy controls. Altogether, the different miRNA profiles in patients with NMOSD and CIS/RRMS further support the notion that NMOSD and CIS/RRMS represent different diseases.
The different miRNA expression levels in patients with NMOSD and CIS/RRMS obtained in the whole blood NGS study could be confirmed by qRT-PCR validation for 9 out of 10 exemplarily chosen miRNAs, 7 of which (including hsa-miR-15b-3p) reached statistical significance. Findings from NGS were thus reproducible by a different method, underscoring the validity of the NGS results. Nevertheless, the absolute fold differences of the top ranking miRNAs between the different groups in the NGS and qRT-PCR studies were only about threefold, and the standard deviations between groups overlapped. Further studies in independent cohorts will therefore be mandatory to corroborate the value of the miRNAs identified in this work as diagnostic biomarkers for NMOSD.
In addition to their potential role as biomarkers, the miRNAs found to be deregulated in NMOSD could be of relevance in the pathophysiology of NMOSD. An in silico enrichment analysis demonstrated, for both comparisons NMOSD vs. CIS/RRMS and NMOSD vs. controls, a highly significant enrichment of the miRNAs deregulated in NMOSD in immune cells (CD15
+, CD14
+, CD56
+, CD19
+, and CD3
+ cells) of relevance for NMOSD. While enrichment in these cells was not unexpected as we studied whole blood containing immune cells, quite remarkably, for both comparisons, the most significant enrichment was observed for CD15
+ cells, i.e., neutrophils and eosinophils [
39,
40]. In contrast to MS, neutrophils and eosinophils are frequently found in the cerebrospinal fluid of patients with NMOSD and are known to play an important role in the pathogenesis of NMOSD lesions [
41‐
43]. Our findings are thus consistent with an altered function of neutrophils and eosinophils in NMOSD and suggest that miRNAs may be involved in the pathophysiology of neutrophils/eosinophils in NMOSD. However, as the functional effects of the miRNAs with altered expression profiles in NMOSD are just partially understood, the precise molecular mechanisms underlying a potential role of these miRNAs in NMOSD and in the regulation of neutrophils/eosinophils remain to be explored.
Limitations of this work are the limited number of patients with NMOSD in the whole blood NGS study and a possible confounding role of immunotherapy in NMOSD patients. However, the results obtained in the whole blood NGS study could clearly be confirmed in a larger number of patients with NMOSD in the qRT-PCR validation study, suggesting that findings obtained in a smaller number of patients are reproducible in larger cohorts. Furthermore, clinical characteristics of the patients included in this study could influence miRNA expression levels, too. While this study was not specifically designed to investigate the association of clinical characteristics with miRNA expression levels, exploratory analyses did not show an association of disease duration, the number of previous relapses, and the time since last relapse with whole blood miRNA expression levels in patients with CIS/RRMS (clinical data available for n = 44 patients) or NMOSD (n = 11).
Among the strengths of this study is the application of NGS technology, which in contrast to more traditional methods of miRNA profiling (miRNA microarrays and qRT-PCR), that are necessarily restricted to a limited number of miRNA targets, has the advantage of allowing for the unbiased detection of all known and even novel miRNAs. Thus, NGS is currently one of the most comprehensive methodological approaches to determine miRNA expression profiles [
44,
45]. Another strength is that
p values were rigorously adjusted for multiple testing, which reduces the risk of false positive findings inherent to the high number of statistical comparisons typically performed in miRNA profiling studies [
44].
Competing interests
The work of SJ has been indirectly supported by research grants from the Dietmar-Hopp-Stiftung and Merck Serono to the Department of Neurology, University of Heidelberg, Germany.
JBS has received speaking fees and travel grants from Bayer Healthcare, sanofi-aventis/Genzyme, and Teva Pharmaceuticals.
FP has received research support as well as speaking fees and travel grants from Bayer Healthcare, Biogen Idec, Merck Serono, sanofi-aventis/Genzyme, Teva Pharmaceuticals, MedImmune, Chugai, Alexion, and Novartis; and is supported by the German Ministry of Education and Research (BMBF/KKNMS, Competence Network Multiple Sclerosis), the Guthy Jackson Charitable Foundation, and the National MS Society of the USA.
KR has received research support from Novartis as well as speaking fees and travel grants from the Guthy Jackson Charitable Foundation, Bayer Healthcare, Biogen Idec, Merck Serono, sanofi-aventis/Genzyme, Teva Pharmaceuticals, and Novartis; and is supported by the German Ministry of Education and Research (BMBF/KKNMS, Competence Network Multiple Sclerosis).
All other authors declare that they have no competing interests.
Authors’ contributions
AK, PL, EM, JBS, FP, FCP, and KR conceived of the study, participated in the design of the study, and analyzed the data. AK and KR drafted the manuscript. CB and AK performed the statistical analysis. LR, JRB, CP, KW, RMG, and FCP acquired and analyzed data. JH and BM performed the serum NGS experiments and analyzed data. SJ analyzed data and revised the manuscript for important intellectual content. All authors read and approved the final manuscript.