Circulating microRNAs as biomarkers in Multiple Sclerosis

Multiple Sclerosis
Based on pathological hallmarks, Multiple Sclerosis (MS) can be subdivided in 4
phases: 1) a pre-clinical phase, 2) relapsing/remitting MS (RRMS), 3) secondary
progressive MS (SPMS), and 4) a burnt-out phase. During RRMS the immune system
is responsible for the pathology, while during SPMS neuro-degeneration is the
predominant pathological factor. It is important to distinguish these two
stages as during the first inhibition of inflammation is a viable treatment
option, while this has no proven efficacy during the latter. Presently there
are no biomarkers available to discern these two phases. Interestingly, 10-15%
of MS patients are diagnosed with another form of MS, called primary
progressive MS (PPMS), without evidence for a preceding relapsing/remitting
phase. As it is conceivable that the biomarker profile of PPMS resembles that
of SPMS, this will be investigated as well.

Genetics of MS
MS is a multifactorial disease with a strong genetic component. A recent
high-profile study describes >50 genetic variants associated with MS. Most of
these affect genes encoding proteins that are known to play a role in
immunity-related cytokine pathways, co-stimulatory pathways, and signal
transduction pathways. Surprisingly, only two genes could be associated with
neuro-degeneration. Interestingly, several genetic variants associated with
autoimmune diseases cannot be pinpointed to protein-encoding genes but localize
to regions containing non-coding RNAs (ncRNAs) or to regulatory regions
containing putative binding sites for transcription factors or ncRNAs (e.g.
miRNA binding sites). Moreover, in some cases the localisation of these disease
susceptibility markers suggests that they affect the expression or sequence of
non-coding RNAs, rather than that of protein-encoding genes.

Circulating miRNAs as biomarkers for MS
A recently identified class of small ncRNAs, miRNAs, can provide novel
biomarkers for MS. miRNAs are small (~19 to 24 nucleotides in length) ncRNAs
that are stable and detectable in different body fluids, including plasma and
cerebrospinal fluid (CSF). The biological function of miRNAs is to fine-tune
gene expression and they do so by inducing the degradation of target mRNAs or
by inhibition of translation of these. Excitingly, circulating miRNA profiles
are disease and disease-stage specific.

The role of miRNAs in MS
A recent review summarizes several studies in which miRNAs have been profiled
in MS lesions or in whole blood. From this a shortlist of miRNAs has emerged
that have been identified in multiple studies. All of the MS miRNA profiles
published to date were obtained by array- or PCR technology and none of them
have investigated circulating miRNAs. We will profile circulating miRNAs using
next generation sequencing (NGS) technology, providing unprecedented
quantification accuracy and sequence depth. Moreover, in contrast to other
techniques NGS allows for identification of novel miRNAs.

Research questions
Although widely studied in cancer research the concept of using circulating
miRNAs as biomarkers for autoimmune diseases is relatively new. Presently, no
papers have been published on MS-specific circulating miRNA profiles (nor on
using the extremely sensitive NGS approach to detect MS-specific miRNA
profiles). It is unknown to date whether CSF or plasma provides the best source
of MS stage-specific miRNA profiles. We will investigate this in the proposed
study.

Primary Objective:
The primary aim of this proposal is to identify miRNA profiles that can be used
as novel MS biomarkers and to design a test, based on these profiles, that can
be used in clinical practice.

Key objectives are:
(1) deep-sequencing of circulating miRNAs in plasma and CSF of MS patients with
relapsing disease or progressive disease,
(2) quantification of known and novel miRNAs as performed previously,
(3) correlation of miRNA expression profiles with the investigated MS stages,
and
(4) the design and validation of a test for the aforementioned MS stages.

In the explorative phase of the study circulating miRNAs will be profiled in
samples already obtained from the *Nederlandse Hersenbank (NHB)* (Netherlands
Brain Bank). Plasma and CSF of MS patients diagnosed with RRMS (n=30), SPMS
(n=30), PPMS (n=10; the number of patients with PPMS is relatively limited in
the NHB), and control samples (non-MS patients; n=30) have been obtained. In
total this study design adds up to 200 paired samples of plasma and CSF.

The technical steps include:
• RNA collection from the samples using the commercially available miRNA
isolation kit of choice (see section 6 of this proposal). These methods are
described in literature; most researchers follow the instructions provided by
the manufacturer (with small modifications).
• Preparation of miRNA sequencing libraries using the respective kit available
from Illumina and subsequent deep sequencing performed on the HiSeq 2000
sequencer (Illumina).
• Analysis of the sequencing data (as published) and determination of stage
specific miRNAs profiles.

Currently, it cannot be predicted whether a single miRNA will be enough to
distinguish between the two MS stages under. It is just as likely that a panel
of miRNAs will be needed to obtain sufficient specificity. Using the stage
specific profiles predictive models will be developed based on stringent
criteria.

This METc application is only relevant to the validation phase of the study:
In the validation phase of the project the profiles selected in the explorative
phase will be validated and for this purpose we will use commercially available
validated single-miRNA PCR assays for the miRNAs of interest. The *MS-centrum
Noord Nederland* (Northern Netherlands MS-center) is an initiative from the
University Medical Center and the Martini Hospital, both in Groningen. Within
the center ~800 patients are regularly visiting the outpatient department of
the two hospitals Of these, 200 RR-MS patients will be sampled and followed
clinically in time. It can be estimated that ~10% of these patients will
convert to SPMS within the next 2-3 years. The predictive value of the
stage-specific miRNA-profiles will be investigated in two different ways.
First, as the samples obtained from the NHB have been obtained over the past
decade, it is essential to investigate whether the profiles found in this
material are also indicative of disease-stage in freshly obtained samples from
an independent cohort of MS patients. To study this the test will be applied to
plasma obtained from 30 RRMS, 30 SPMS and 30 PPMS patients from Groningen,
thereby measuring only a limited set of miRNAs that are representative for the
disease-stage profile.
Secondly, we will also apply the test to samples collected twice a year from
~20 converted patients that were obtained before and after conversion and
compare these to controls that did not convert from RRMS to SPMS during this
period. Conversion to SPMS from RRMS will be defined retrospectively as
confirmed progression of disability without concomitant relapses.

This study only involves routine venapuncture and study related adverse events
are therefore not anticipated.
Individual participants will not benefit from participating in this study.