Circulating miR-150 in CSF is a novel candidate biomarker for multiple sclerosis

Abstract

Objective: To explore circulating microRNAs (miRNAs) in cell-free CSF as novel biomarkers for multiple sclerosis (MS).

Methods: Profiling of miRNAs in CSF of pooled patients with clinically isolated syndrome (CIS), patients with relapsing-remitting MS, and inflammatory and noninflammatory neurologic disease controls was performed using TaqMan miRNA arrays. Two independent patient cohorts (n = 142 and n = 430) were used for validation with real-time PCR.

Results: We reliably detected 88 CSF miRNAs in the exploratory cohort. Subsequent validation in 2 cohorts demonstrated significantly higher levels of miR-150 in patients with MS. Higher miR-150 levels were also observed in patients with CIS who converted to MS compared to nonconverters, and in patients initiating natalizumab treatment. Levels of miR-150 correlated with immunologic parameters including CSF cell count, immunoglobulin G index, and presence of oligoclonal bands, and with candidate protein biomarkers C-X-C motif chemokine 13, matrix metallopeptidase 9, and osteopontin. Correlation with neurofilament light chain (NFL) was observed only when NFL was adjusted for age using a method that requires further validation. Additionally, miR-150 discriminated MS from controls and CIS converters from nonconverters equally well as the most informative protein biomarkers. Following treatment with natalizumab, but not fingolimod, CSF levels of miR-150 decreased, while plasma levels increased with natalizumab and decreased with fingolimod, suggesting immune cells as a source of miR-150.

Conclusions: Our findings demonstrate miR-150 as a putative novel biomarker of inflammatory active disease with the potential to be used for early diagnosis of MS.

Classification of evidence: This study provides Class II evidence that CSF miR-150 distinguishes patients with MS from patients with other neurologic conditions.

Figures

Figure 1. Levels of miR-150 are elevated in patients with multiple sclerosis (MS) and patients with clinically isolated syndrome (CIS) who convert to MS

Relative levels of mature microRNAs (miRNAs) were measured using multiplexed specific TaqMan miRNA assays and normalized to an average of 3 spike-ins (cel-miR-39, cel-miR-54, and cel-miR-238). Levels of (A) miR-150 are significantly different between disease groups, while (B) there was no difference between levels of miR-145. Levels of miR-150 are also (C) higher in patients with CIS who converted to MS (CIS-MS) compared to those who never converted and (D) can discriminate MS from noninflammatory neurologic disease controls (NINDCs) based on receiver operating characteristic (ROC) curve analysis. Graph intersection indicates the cutoff value for miR-150 that proved the best specificity and sensitivity. Levels of miR-150 in relation to descriptive disease parameters (E) oligoclonal bands (OCB), (F) relapse and remission, (G) number of MRI lesions, and (H) subsequent treatment with natalizumab. Lines in dot plots represent median and interquartile range. *p < 0.05, **p < 0.01, ***p < 0.001. INDC = inflammatory neurologic disease controls.

Figure 2. Levels of miR-150 are altered in CSF and plasma following treatment with disease-modifying drugs

Relative levels of mature miR-150 were measured in CSF and in plasma from patients with multiple sclerosis treated with (A,B) natalizumab and (C,D) fingolimod at baseline and at 12 months. Quantification was performed using multiplexed specific TaqMan microRNA assays and normalized to an average of 3 spike-ins (cel-miR-39, cel-miR-54, and cel-miR-238). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3. MicroRNA (miRNA) pairs can be used to normalize miRNA levels

The ratio of miR-150 and miR-204, calculated and normalized using the ΔΔCT method (see Methods), is significantly different (A) between disease groups and (B) between patients with clinically isolated syndrome (CIS) who have converted or not converted to multiple sclerosis (MS). The miR-150/miR-204 ratio improves (C) the area under receiver operating characteristic (ROC) (AUC) curve, indicating improved specificity and sensitivity to discriminate MS from noninflammatory neurologic disease controls (NINDC) compared to miR-150 normalized to spike-ins. The miR-150/204 ratio can furthermore (D) predict conversion from CIS to MS as indicated by the ROC curve generated to compare converters and nonconverters. The miR-150/miR-204 ratio is an independent significant (p = 0.017) predictor of conversion when known risk factors such as age, oligoclonal bands (OCB), and MRI lesions are taken into account, and it can improve their combined predictive value. Lines represent (A, B) median and interquartile range. **p < 0.01, ***p < 0.001. INDC = inflammatory neurologic disease controls.