WO2010136232A1 - In vitro method suitable for patients suffering from cis for the early diagnosis or prognosis of multiple sclerosis - Google Patents

Abstract

In vitro method suitable for patients suffering from CIS for the early diagnosis or prognosis of multiple sclerosis. The method comprises the identification of the up- regulation of the CHI3L1 protein or gene in CSF samples isolated from those patients suffering from CIS. Furthermore the invention refers to a method for screening or identifying compounds or molecules active for treating those patients suffering from MS, to kits for the early diagnosis of MS or for the prognosis of MS disability progression in patients suffering from CIS and to a pharmaceutical composition comprising a therapeutically effective amount of an agent, active for inhibiting the activity of the CHI3L1 protein or the expression of the CHI3L1 gene.

Description

IN VITRO METHOD SUITABLE FOR PATIENTS SUFFERING FROM CIS FOR THE EARLY DIAGNOSIS OR PROGNOSIS OF MULTH¹LE

SCLEROSIS

FIELD OF THE INVENTION

The present invention refers to in vitro methods for the early diagnosis of Multiple Sclerosis (MS) or for the prognosis of the MS progression and/or the development of disability, suitable for patients suffering from Clinically Isolated Syndrome (CIS).

Said methods comprise the in vitro analysis of the CHI3L1 gene expression level (by means of either the measuring of the transcribed mRNA levels of the CHI3L1 gene, or its complementary cDNA, or also the CHI3L1 protein expression level), in samples preferably isolated from the cerebrospinal fluid (CSF) of those patients suffering from CIS. Furthermore, the present invention refers to a method for screening and identifying compounds or molecules active for inhibiting the progression from CIS to clinically definite MS (CDMS) in patients suffering form CIS.

Therefore, the present invention can be encompassed in the medical field, more specifically in the field covering neurodegenerative diseases.

STATE OF THE ART

MS usually initiates with an acute or subacute episode of neurological disturbance known as CIS. However, on the contrary, not every patient suffering from CIS ends developing MS disease. At that stage, magnetic resonance imaging (MRI) is an important tool both for predicting the conversion to CDMS and the development of disability (Brex et al., 2002; Tintore et al., 2006; Fisniku et al, 2008). In addition, the presence of oligoclonal bands (OB) was found to be a risk factor for conversion to MS independently of baseline MRI (Tintore et al., 2008). However, with the exception of

OB, the role of other biomarkers in CIS patients for predicting development of CIS pathological state to MS disease is controversial (Berger et al., 2003; Pelayo et al., 2007; Kuhle et al., 2007).

On the other hand, proteomics is well suited for biomarker discovery, and mass spectrometry-based approaches have been widely applied for protein identification. Proteomic studies are rapidly emerging in MS patients (Hammack et al., 2003; Dumont et al., 2004; Noben et al., 2005, Irani et al., 2006; Lehmensiek et al., 2007; D'Aguanno et al., 2008; Stoop et al., 2008; Chiasserini et al., 2008; Qin et al., 2009) and have helped to identify proteins that may be potential disease-specific markers.

iTRAQ is a non-gel based technique used to identify and quantify proteins from different sources in one single experiment. It uses isotope coded covalent tags. iTRAQ is used in proteomics to study quantitative changes in the proteome. The method is based on the covalent labeling of the N-terminus and side chain amines of peptides from protein digestions with tags of varying mass. There are currently two mainly used reagents: 4-plex and 8-plex, which can be used to label all peptides (In theory) from different samples/treatments. These samples are then pooled and usually fractionated by nano liquid chromatography and analyzed by tandem mass spectrometry. A database search is then performed using the fragmentation data to identify the labelled peptides and hence the corresponding proteins. The fragmentation of the attached tag generates a low molecular mass reporter ion that can be used to relatively quantify the peptides and the proteins from which they originated, using software such as the freely available i-Tracker.

Therefore, the finding of validated biomarkers associated with the conversion to MS at the time of a CIS event would be nowadays of utmost importance. Biomarker discovery in CIS patients at the time of disease onset will be useful (i) to differentiate CIS patients that will convert to MS from CIS patients who develop disorders other than MS, (ii) as prognostic factors for MS disease progression and development of disability, and (iii) to better understand the pathogenesis underlying the early stages of the disease. So, the present invention is focused on providing validated biomarkers by carrying out the analysis of the proteome profile present in cerebrospinal fluid (CSF) samples from patients with CIS who will develop MS, patients with CIS who will not convert to MS, and patients with other inflammatory and non-inflammatory neurological disorders. Its proximity to inflammatory lesions in the central nervous system (CNS) makes CSF ideal for the identification of biomarkers related to the underlying disease.

SUMMARY OF THE INVENTION

Brief description of the invention The main objective of the present invention was the identification of biomarkers, at the time of a CIS event, associated with conversion to CDMS. Therefore, the present invention provides an in vitro method, suitable for patients suffering from CIS, for the early diagnosis of MS, or for the prognosis of MS disability progression, that comprises the analysis of the CHI3L1 gene expression level (by means of either the measuring of the transcribed mRNA levels of the CHI3L1 gene, or its cDNA, or also the CHI3L1 protein level), in samples preferably isolated from the cerebrospinal fluid (CSF) of those patients suffering from CIS.

The nucleotide sequence of the CHI3L1 gene has been entered in the NCBI database (ID: 1116): Chitinase 3-like 1 (cartilage glycoprotein-39).

Any conventional kit assay can be used in the framework of the invention for the early diagnosis of MS or for the prognosis of CDMS disability progression, both in patients suffering from CIS, provided that it is able to carry out the in vitro analysis of the CHI3L1 gene expression level (by means of either the measuring of the transcribed mRNA levels of the CHI3L1 gene, or its cDNA, or also the CHI3L1 protein level), in samples preferably isolated from the cerebrospinal fluid (CSF) of those patients suffering from CIS.

In a preferred embodiment the CHI3L1 gene expression level is evaluated by means of the CHI3L1 protein level and is considered to be up-regulated when this level is up to the cut-off value of 287.9 mg/ml in samples of CSF. In a first screening phase, mass spectrometry-based proteomic approach iTRAQ (isobaric tag for relative and absolute quantisation) was applied to pooled CSF samples from CIS patients that will convert to CDMS and patients who will remain as CIS. Proteins associated with conversion to CDMS were identified in pooled CSF samples from patients with CIS that converted to CDMS (Group 2) and patients who will remain as CIS (Group 1).

In a second phase of validation, the most represented differentially expressed proteins were selected for validation in individual CSF samples of two groups of patients pertaining to different populations by ELISA. Only CHI3L1 (also known as YKL40) was validated. CSF levels for this protein were found increased (or up-regulated) in CIS patients that will convert to CDMS (Group 2), as compared with CIS patients who will continue as CIS (Group 1), and also with CIS patients that develop other neurological disorders different to MS (Control group). Interestingly, high baseline CSF CHI3L1 levels were associated with disability progression during follow-up and shorter time to pass from CIS to CDMS.

The following flow chart (Scheme I) summarizes the different steps undertaken in the present invention to reach the proposed results. In a first phase of the study (screening phase), a mass spectrometry-based proteomic approach (iTRAQ) was applied to pooled CSF samples from 30 CIS patients who converted to CDMS and 30 CIS patients who remained as CIS (original CIS cohort) in order to identify proteins associated with conversion to MS. In a second phase of the study (validation phase), three proteins, CHI3L1, VDBP (vitamin D binding protein) and CP (ceruloplasmin), were selected for validation in individual CSF samples by means of ELISA. CSF levels for these proteins were measured in a partially overlapping CIS cohort comprised of 48 CIS patients who converted to MS and 36 CIS patients who continued as CIS (first validation cohort). Only CHI3L1 was validated and an additional analysis was performed in order to evaluate the association between CSF levels of CHI3L1 and the progression of CIS patients to CDMS. Finally, CSF CHI3L1 levels were determined in a totally independent CIS cohort comprised of 26 CIS patients who converted to MS and 26 CIS patients who remained as CIS (second validation cohort). CHBL 1 was also validated in this new CIS cohort. The follow-up time (FT) and the percentage of new patients included in the different CIS cohorts are indicated:

Scheme I

Therefore, the results obtained in the present invention clearly show that patients who will convert to CDMS may be distinguished from those patients that will remain as

CIS by means of the analysis of the CHI3L1 gene expression level (by means of either the measuring of the transcribed mRNA levels of the CHI3L1 gene, or its cDNA, or also the CHBLl protein level), in samples preferably isolated from the cerebrospinal fluid (CSF) collected at the time of a CIS event. Moreover, the present invention provides prognostic biomarkers for conversion to CDMS, and development of disability, which may help to better understand the etiopathogenesis of early stages of

MS. Due to the high levels of CHI3L1 found in CSF samples isolated from CIS patients which develop to CDMS, it may be assumed that the CHI3L1 is a therapeutic target, suitable for inhibiting the progression from CIS to CDMS in patients suffering from CIS that have the CHI3L1 gene up-regulated. Therefore, the present invention further refers to a method for screening and identifying compounds or molecules active for inhibiting the progression from CIS to CDMS in patients suffering form CIS that have the CHI3L1 gene up-regulated. Compounds or molecules active for treating CIS patients that will likely develop to MS, as described in the present invention, refers to compound or molecules able to cure the disease by means of avoiding the transfer from CIS state to CDMS state.

Description of the figures

Figure 1. It shows the CSF (above) and serum (below) levels of CHI3L1 protein (y- axis) in CIS patients and controls included in the validation phase of the study. Departing from the left column of the x-axis, the first column represents the control group, the second column represents the Group 1 and the third column represents the Group 2. Levels of CHI3L1 protein (mg/ml) were determined by ELISA in CSF and serum samples from CIS patients that did not convert to CDMS (Group 1 , n=48), CIS patients that converted to CDMS (Group 2, n=36), and individuals with other neurological disorders (control group, n=20). CSF CHI3L1 levels were significantly higher in Group 2 compared to Group 1 and controls (*p=0.1 x 10^"4 and p=0.012 respectively, Mann Whitney U test). Serum CHI3L1 levels were higher in CIS patients compared with controls (Group 1 vs. controls, *p=0.048; Group 2 vs. controls, *p=0.003). Boxes represent the 75th and 25th percentiles divided horizontally by the median. Whiskers are drawn to the nearest value not beyond a standard span (1.5 and 3 x the interquartile range) from the 75th and 25th percentiles. VDBP: vitamin D binding protein.

Figure 2. Kaplan-Meier curves (percent survival vs time) for time to CDMS according to baseline CSF levels of CHI3L1 in patients with CIS. Levels of CHI3L1 were categorized into low and high as described in results. CIS patients with high CHI3L1 levels had a significantly shorter time to develop to CDMS compared to patients with low CHI3L1 (log-rank p value= 0.003). Median survival time (95% confidence interval) in patients with high and low levels of CHI3L1 in CSF is 17.6 months (11.6 - 23.7) and 34.2 months (16.9 - 51.5) respectively.

Detailed description of the invention

The present invention provides an in vitro method for the early diagnosis of MS or for the prognosis of MS disability progression, in patients suffering from CIS, comprising:

• the analysis of the CHI3L1 gene expression level (by means of either the measuring of the transcribed mRNA levels of the CHI3L1 gene, or its complementary cDNA, or also the CHI3L1 protein expression level), in samples preferably isolated from the cerebrospinal fluid (CSF) of those patients suffering from CIS, and

• the comparison of the said CHI3L1 gene expression level detected in a sample of the individual suffering from CIS with the corresponding CHI3L1 gene expression level detected in the samples of control individuals or in earlier samples of the same individual or with normal reference values.

The method provided by the present invention has high sensitivity and specificity, and is based on subjects or individuals diagnosed first with CIS and later on with MS, having the CHI3L1 gene expression up-regulated, in comparison with subjects having other neurological diseases different from MS, formerly diagnosed with CIS.

Any conventional assay can be used in the framework of the invention for the early diagnosis of MS, or for the prognosis of MS disability progression, in patients suffering from CIS, provided that it is able of measuring in vitro the CHI3L1 gene expression level present in samples collected from the individuals to be analyzed and from control individuals. When the concentration of CHI3L1 protein is the CHI3L1 gene expression parameter to be measured, the method of the invention comprises a first step which places in contact either directly a sample containing CHI3L1 protein or an extract containing said protein isolated thereof, with a composition of one or more specific antibodies against one or more epitopes of the CHDLlprotein, and a second step for quantifying the complexes formed by those antibodies and the CHDLlprotein.

There is a wide variety of immunological assays available for detecting and quantifying the formation of specific antigen-antibody complexes; a number of competitive and non-competitive protein binding assays have been described in the state of the art, and a large number of these assays are commercially available. Therefore, the CHI3L1 protein can be quantified with antibodies such as, for example: monoclonal antibodies, polyclonal antibodies, either intact or fragments thereof, "combi-bodies" and Fab or scFv fragments of antibodies specific against the CHI3L1 protein; these antibodies being human, humanized or of a non-human origin. The antibodies used in these assays can be marked or unmarked; the unmarked antibodies can be used in agglutination assays; the marked antibodies can be used in a wide variety of assays. The marking molecules that can be used to mark the antibodies include radionucleotides, enzymes, fluorophores, chemiluminescent reagents, enzymatic substrates or cofactors, enzymatic inhibitors, particles, dyes and derivatives.

There is a wide variety of well known assays which can be used in the present invention using non-marked antibodies (primary antibody) and marked antibodies (secondary antibody); these techniques include Western blot, ELISA (Enzyme-Linked Immunosorbent assay), RIA (Radioimmunoassay), competitive EIA (Competitive enzyme immunoassay), DAS-ELISA (Double antibody sandwich-ELISA), immunocytochemical and immunohistochemical techniques, techniques based on the use of protein biochips or microarrays including specific antibodies or assays based on colloidal precipitation in formats such as dipsticks. Other ways of detecting and quantifying the protein CHI3L1 include affinity chromatography techniques, ligand binding assays or lectin binding assays. The preferred immunoassay in the method of the invention is ELISA assay. Any antibody or combination of antibodies, specific against one or more epitopes of the CHBLl protein can be used in this immunoassay. As an example of one of the many possible formats of this assay, a monoclonal or polyclonal antibody, or a fragment of this antibody, or a combination of antibodies, which coat a solid phase are placed in contact with the sample to be analyzed and are incubated for a suitable time and in suitable conditions for forming the antigen-antibody complexes. After a washing in suitable conditions to eliminate the non-specific complexes, an indicator reagent, comprising a monoclonal or polyclonal antibody or a fragment of this antibody, or a combination of these antibodies, bonded to a signal generating compound is incubated with the antigen-antibody complexes under suitable conditions and for a suitable time.

The presence of the CHI3L1 protein in the sample to be analyzed is detected and quantified, should it exist, by measuring the generated signal. The amount of CHBL 1 protein present in the sample to be analyzed is proportional to that signal.

In a first screening phase, mass spectrometry-based approach was applied in order to identify proteins associated with conversion to CDMS in pooled CSF samples from patients with CIS that converted to CDMS and patients who will remain as CIS during the follow-up period.

In a second phase, selected proteins were validated by an independent technique in individual CSF samples taken from two groups of CIS patients pertaining to different populations. The data obtained in the present invention show that the CSF proteome profile is different between CIS patients that will convert to MS and CIS patients who will not convert, and propose candidate markers associated with conversion to MS in CIS patients.

One of the recent methodologies developed by Applied Biosystems has been called iTRAQ, which allows a relative and absolute quantification of the proteins present in samples from different disease stages. That methodology was used in the present invention to identify proteins associated with conversion to MS in CIS patients classified under stringent criteria in a first phase of screening. Interestingly, patients who will convert to CDMS could be distinguished from those patients that will remain as CIS by applying said proteomic approach to CSF samples collected at the time of a CIS event.

The most represented proteins that were differentially expressed in the pool comparisons were selected for a first and second phase of validation in individual CSF samples taken from two groups of CIS patients pertaining to different populations using a different analytical approach. To give more strength to the validation process, approximately 50% of the CSF samples used for validation came from new CIS patients not included in the initial CSF pools. Only CHI3L1 was finally validated by ELISA.

CIS patients were classified into two groups (high/low) based on a cut-off value of CHI3L1 of 287.9 mg/ml measured in CSF samples. This cut-off value was calculated from the mean CSF CHI3L1 levels + 2 standard deviations obtained from the group of CIS patients with developed other neurological disorders different to MS (control group). The CHI3L1 protein is considered to be up-regulated or down-regulated when its level is up or down to said cut-off value, respectively.

In 5 out of 6 pool comparisons, CHI3L1 was overexpressed in CIS patients who will convert to CDMS. Interestingly, proteomic results were confirmed by ELISA, and CSF CHI3L1 levels were found increased in this group of CIS patients. The fact that both validation cohorts included less stringent classification criteria of conversion to MS compared to the original cohort used in the screening phase, i.e. number of BC (Barkhof criteria) at baseline MRI (magnetic resonance imaging) and follow-up times, indicates that CHI3L1 can be applied to a broad spectrum of CIS patients to discriminate between patients who will later convert to MS and patients who will continue as CIS.

The present invention shows that CHI3L1 levels correlated in CSF samples with baseline MRI abnormalities that reflect the degree of brain inflammation and lesion burden, and with development of disability progression at follow-up. Interestingly, high baseline CSF protein levels give rise to a poorer prognosis, as they were associated with shorter time to CDMS.

When mRNA or cDNA are the parameters used for the quantification of CHI3L1 gene expression the invention has various steps. Firstly, once the sample is obtained and the total RNA is extracted, the method of the invention, for the detection either of mRNA or of the corresponding cDNA of the CHI3L1 gene, comprises a first step of amplifying the mRNA present in the total RNA extract, or the corresponding cDNA synthesized by reverse transcription of the mRNA, and a second step of quantifying the amplification product of the mRNA or cDNA of the CHI3L1 gene.

An example of amplifying the mRNA consists of retrotranscribing the mRNA into cDNA (RT), followed by polymerase chain reaction (PCR); PCR is a technique for amplifying a certain nucleotide sequence (target) contained in a mixture of nucleotide sequences. An excess pair of oligonucleotide primers which hybridize with the complementary strands of the target nucleotide sequence is used in the PCR. Then an enzyme with polymerase activity (DNA Taq Polymerase) extends each primer using as a template the target nucleotide sequence. The extension products are then converted into target sequences after disassociation of the original target strand. New primer molecules hybridize and the polymerase extends them; the cycle is repeated to exponentially increase the number of target sequences. This technique is described in patents US 4,683,195 and US 4,683,202. Many methods for detecting and quantifying PCR amplification products have been previously described and any of them can be used in this invention.

In another example, the detection of the mRNA is carried out by transferring the mRNA to a nylon membrane by means of transfer techniques such as for example Northern blot, and detecting it with specific probes of the mRNA or the corresponding cDNA of the CHI3L1 gene. In a particular embodiment, the amplification and quantification of the mRNA corresponding to the CHDL 1 gene is carried out at the same time by means of realtime quantitative RT-PCR (Q-PCR).

The last step of the method of the invention for diagnosing in vitro MS comprises comparing the amount of CHI3L1 protein, the amount of CHI3L1 mRNA or the amount of the corresponding cDNA in a sample from an individual, with the amount of CHI3L1 protein, the amount of CHI3L1 mRNA or the amount of the corresponding cDNA, detected in samples of control subjects or in earlier samples of the same individual, or with normal reference values.

The invention also provides an in vitro method for screening compounds or molecules active for inhibiting the progression from CIS to CDMS, in patients suffering from CIS, that comprises:

• placing a culture of cells isolated from the samples (preferably CSF samples) obtained from CIS patients, in contact with the candidate compound, under the suitable conditions and for the suitable time to allow them to interact, • detecting and quantifying the expression levels of the CHI3L1 gene by measuring either CHI3L1 protein, CHI3L1 mRNA or cDNA and • comparing said expression levels with those of the control cultures of those cells not treated with the candidate compound.

The quantification of the expression levels of the CHI3L1 gene is carried out similarly to that indicated in the method of the invention for detecting in vitro the presence of MS in an individual.

When an agent reduces the CHI3L1 gene expression levels or reverses the effects of the high expression of said gene, this agent becomes a candidate for MS therapy.

Therefore, another object of the invention refers to the use of nucleotide sequences derived from the CHI3L1 gene or of the peptides encoded thereof in methods of finding, identifying, developing and evaluating the effectiveness of compounds for MS therapy. It is essential to point out the importance that it has recently acquired drug screening methods based on the competitive or non-competitive binding of the potential drug molecule to the therapeutic target, in drug discovery.

Another additional object of the invention refers to the use of nucleotide sequences derived from the CHI3L1 gene or peptides encoded thereof for the in vitro diagnosis of MS, for determining the stage or severity of said MS in the individual, or for monitoring the effect of the therapy administered to an individual having MS.

Another object of the invention consists of providing agents characterized in that they inhibit the expression of CHI3L1 gene and/or activity of the CHI3L1 protein. These agents, which can be identified and evaluated according to the present invention, can be selected from the group formed by:

• an antibody, or combination of antibodies, specific against one or more epitopes present in the CHI3L1 protein, preferably a human or humanized monoclonal antibody; also being able to be a fragment of the antibody, a single-chain antibody or an anti-idiotype antibody,

• peptides, phosphopeptides, anti-sense molecules, ribozymes, siRNAs, triple helix molecules, etc., inhibiting the expression of CHI3L 1 gene and/or activity of the CHI3L1 protein, and

• antagonist compounds of the CHI3L1 protein, inhibiting one or more of the functions of the CHI3L1 protein.

A pharmaceutical composition comprising a therapeutically effective amount of one or several of the previously mentioned agents together with one or more excipients and/or carrier substances also constitutes an object of the present invention. Furthermore, said composition may contain any other active ingredient that does not inhibit the function of the CHI3L1 protein.

The excipients, carrier substances and auxiliary substances must be pharmaceutically and pharmacologically tolerable, such that they can be combined with other compounds of the formulation or preparation and do not have any adverse effects on the treated organism. The pharmaceutical compositions or formulations include those which are suitable for oral or parenteral administration (including subcutaneous, intradermal, intramuscular and intravenous), although the best administration route depends on the patient's condition. The formulations can be in the form of single doses. The formulations are prepared according to known methods in the field of pharmacology. The amounts of active substances to be administered may vary according to the particularities of the therapy.

A further aspect of the present application consists of a diagnostic kit for carrying out the present invention. Therefore, in a particular embodiment, the present invention includes a kit comprising an antibody especially recognizing the CHBLl protein and a carrier in a suitable container. In another particular embodiment, this kit is used for the early diagnosis of MS or for the prognosis of MS disability progression in patients suffering from CIS, or for monitoring the effect of the therapy administered to an individual having MS.

A final aspect of the present invention consists of an in vitro method for diagnosing the survival time of a patient with MS comprising the evaluation of the CHI3L1 gene expression level in a sample extracted from the patient by means of determining in said sample at least one parameter related to the CHI3L1 gene expression which is selected from the level of its messenger RNA, the concentration of CHI3L1 protein or the enzymatic activity of said protein, and the comparison of the obtained value with the value corresponding to one or more normal tissue samples.

In summary, the approach carried out in the present invention permitted the identification of a specific biomarker, associated with conversion from CIS to MS, which may help to better understand the etiopathogenesis of early stages of MS. This biomarker is CHI3L1 which surprisingly constitutes a strong prognostic marker for disease conversion from CIS to MS and development of MS disability, and as a therapeutic target useful for inhibiting the progression from CIS to CDMS in patients suffering form CIS that have the CHI3L1 gene up-regulated. EXAMPLES

Example 1. Patients

Screening phase

For the screening phase, sixty patients with CIS recruited at the CEM-Cat from 1995 onwards were selected based on the following criteria: Group 1/ No conversion to CDMS, IgG OB negative, and normal brain MRI after 5 years of follow-up (N=30). Group 2/ Conversion to CDMS, presence of OB, and 3 or 4 Barkhof criteria (BC) at baseline (N=30). The study was approved by the Ethics Committee of VaIl d'Hebron University Hospital. Clinical and MRI assessments have been previously described elsewhere (Tintore et al., 2006). Briefly, brain MRI scans were performed at baseline and after 1 and 5 years of follow-up on a 1.0-T or 1.5-T magnet with a standard head coil. MRI included the following sequences: transverse proton-density and T2- weighted conventional spin echo, and contrast-enhanced Tl -weighted spin-echo. The number of BC (Barkhof et al., 1997; Tintore et al., 2008), number of T2 lesions, number of gadolinium enhancing lesions, and number of new T2 lesions were scored. Disability was evaluated according to the Expanded Disability Status Scale (EDSS) score in each visit and only EDSS performed during stability periods were considered. Clinically definite MS (CDMS) was diagnosed when there was a second attack with a new neurological abnormality that was confirmed by examination (Poser et al., 1983). Time of follow-up was computed as the difference between the date of the last visit and the date of the CIS event.

A summary of demographic and clinical characteristics of CIS patients included in the screening phase is depicted in Table 1: (a) Data are expressed as mean (standard deviation); (b) Data are expressed as median (interquartile range). P values were obtained following comparisons between Groups 1 and 2 by means of chi-square test (gender and clinical presentation) and Mann-Whitney's test (remaining variables). TABLE l

First validation phase Eighty- four CIS patients recruited at the CEM-Cat were selected for a first validation phase. Forty-eight patients fulfilled Group 2 criteria, and 20 patients strictly satisfied Group 1 criteria. An additional group of 16 patients with less stringent Group 1 criteria, to be exact, no conversion to CDMS, negative OB, and normal brain MRI after 1 year follow-up was also included. Clinical and MRI assessments in these 84 patients were comparable to those described in the screening phase. Additionally, a control group of 20 individuals with other neurological disorders was included in the study. Demographic and clinical characteristics of CIS patients and controls are shown in Table 2. Eighteen patients (50.0%) from Group 1 and 25 patients (52.1%) from Group 2 were also included in the screening phase of the study.

Demographic and clinical characteristics of CIS patients and controls are shown in Table 2: (a) refers to number and percentage of patients that were also part of the screening phase. Data are expressed as mean (standard deviation) unless otherwise stated. OND: other neurodegenerative diseases. OIND: other inflammatory neurological disorders. Patients with systemic autoimmune disorders included Sjogren's syndrome (n=2), vasculitis (n=2), systemic lupus erythematosus (n=l), neurosarcoidosis (n=l), and Behcet's disease (n=l). ON: optic neuritis. PS: paraneoplastic syndrome. THS: Tolosa-Hunt syndrome. RE: Rasmussen's encephalitis

TABLE 2

Second validation phase

A second independent cohort comprised of 52 new CIS patients recruited at the Hospital Ramόn y Cajal (Madrid, Spain) was used for validation of candidate proteins. Group l_included 26 CIS patients who remained as CIS during the follow-up, with negative OB_and 0 (11 patients; 42.3%), 1 (11 patients; 42.3%), or 2 (4 patients; 15.4%) BC at baseline brain MRI. Group 2 included 26 CIS patients who converted to MS (by the Poser criteria in 24 patients and McDonald criteria in 2 patients; McDonald et al., 2001), with positive OB and 3 or 4 BC at baseline brain MRI. The median time (interquartile range) between the CIS event and CSF extraction was 36.5 days (9.5 - 111.3 days) in patients from Group 1 and 26.0 days (10.0 - 78.8 days) in patients from Group 2. Sixteen individuals with inflammatory neurological disorders were used as controls. Table 2 describes demographic and clinical characteristics of CIS patients and controls included in the second validation cohort.

Example 2. CSF sampling and pooling strategy

CSF samples were collected at baseline by lumbar puncture and centrifuged for 5 min at 1500 rpm to remove cells. Samples were subsequently used for routine CSF diagnostics that included biochemistry and determination of IgG OB by agarose isoelectric focusing combined with immunoblotting and immunoperoxidase staining. The remaining volume of the samples was aliquoted and conserved at -8O⁰C until used. CSF characteristics of patients included in Groups 1 and 2 are shown in Table 1.

A CSF pooling strategy was designed in the screening phase to identify proteins differentially expressed between patients belonging to Groups 1 and 2. Twelve CSF pools were created, 6 per group, each pool containing CSF from 5 different patients, and each patient contributing with 300 μl to a final volume of 1.5 ml. Pools between groups were sex- and age- matched, and patients between pools were different. An illustration of the pooling design is depicted in Scheme II. Thus, Scheme II shows the patient classification and CSF pooling strategy used in the screening phase to identify differentially expressed proteins. Sixty patients with CIS were categorized into two groups (Groups 1 and 2) based on stringent classification criteria. Six CSF pools were generated per group. Each CSF pool contained a total volume of 1.5 ml from 5 different patients, and each patient contributed with an equal volume of 300 μl of CSF:

Scheme II

Group 1 Group 2

* No conversion to MS V Converiion to CDMS

Classification S Normal MRl ater 5 year fbHow-up / 3 or 4 iC criteria

^■f Igβ OB negative ^■f Presence of Ol

Example 3. Proteomic analysis

Scheme in exemplifies the workflow followed for proteomic analysis, which comprised the following steps. Thus, Scheme III shows the workflow followed for proteomic analysis of CSF pools. After sampling and pooling the CSF of CIS patients included in the screening phase of the study, the major proteins (IgG and albumin) were removed using an affinity column kit to amplify the detection rank. The protein content of the samples was then precipitated in ice-cold acetone and quantified using the Bradford's method. In order to perform the iTRAQ technique, all samples were suitably prepared and digested into tryptic peptides. Three independent 4-plex iTRAQ experiments were carried out comparing 2 pools of each of the 2 different groups (Groups 1 and 2). Sample complexity was decreased by fractionation using cation exchange chromatography and then an LC-MS/MS was performed. The information processing was carried out using the Protein Pilot 2.0 software. Finally, after the selection of the most plausible proteins associated with conversion from CIS to CDMS, candidate proteins were validated independently by ELISA to rule out false positives obtained during the screening phase:

Scheme III

CSF sampling and pooling

IgG and albumin depletion

Protein precipitation in acetone

Protein quantif aication (Bradford)

Sample preparation and protein digestion with trypsin

( iTRAQ )

Cex and nanoLC-MS/MS

Protein Pilot

Data analysis of DEP

Individual validation of top candidates by an alternative technique Sample depletion and preparation

After thawing, CSF samples were first concentrated and then albumin and IgG depleted with the ProteoPrep Immunoaffinity Albumin and IgG Depletion Kit (SIGMA-ALDRICH, St. Louis, Missouri, USA) following the manufacturer's recommendations. Subsequently, samples were precipitated by adding 4 volumes of ice-cold acetone overnight, centrϋiiged, and the protein pellet diluted in water. Finally, protein concentration was determined using the Bradford's protein quantification method (Bio-Rad Protein Assay, Bio-Rad Laboratories GmbH, Munich).

JTRAO labelins

CSF pools were analyzed by iTRAQ as follows: Three independent 4-plex experiments were performed, and each experiment contained 2 pools from each group. Fifty μg of protein were transferred to a sample tube and dried in a speedvac. After re- dissolving samples in the Sample Buffer-Plasma following the manufacturer's recommendations (iTRAQTM Reagents Application Kit-Plasma Protocol, Applied Biosystems, Foster City, CA, USA), samples were reduced, alkylated, and enzymatically digested with porcine trypsin (PROMEGA, Madison, WI, USA) as per the manufacturer's protocol. The resulting peptides were then labeled with the iTRAQTM reagents and pooled following the manufacturer's protocol. Due to the complexity of the peptide mixture, a cation exchange chromatography was next carried out. Then, peptides were eluted using salt steps and seven fractions finally collected using a range from 25 mM to IM of KCl.

Nanoflow liquid chromatography and tandem mass spectrometry fnanoLC-MS/MS) analysis Samples were run on a Q-Star Pulsari (Applied Biosystems) instrument fitted with a nano-ESI source, previous nanoLC separation in an Ultimate II system (LCPackings). Prior to LC-MS analysis, tryptic peptide mixtures were desalted and concentrated on RP-C 18 columns (Spec Cl 8, Varian). Fractions were separated in a reverse phase Atlantis dC18 NanoEase Column, 75μm x 150mm (Waters), using a linear 5-55% acetonitrile gradient into 0.1% formic acid over 120 min. An electrospray voltage of 2400 V was used. Data dependent software (Analyst QS 1.1, Applied Biosystems) was employed for online analyses consisting of a survey scan followed by sequential isolation and fragmentation of the three most intense peaks.

Data processing and selection of differentially expressed proteins Protein identification and quantification were performed using the ParagonTM Algorithm in thorough search mode implemented in the ProteinPilotTM Software 2.0. Proteins were identified by searching in the UniProt/Swiss-Prot or NCBInr databases. For the analysis, 6 pool comparisons were performed as follows: pool #1 from Group 1 was directly compared with pool #1 from Group 2, pool #2 from Group 1 was compared with pool #2 from Group 2, and so forth. From each pool comparison, differentially expressed proteins between Groups 1 and 2 (p-values < 0.05) were selected, and the number of pool comparisons in which a selected protein was differentially expressed was counted. Proteins with changing directions in their expression, i.e. upregulated and downregulated in different pool comparisons, were not considered in the analysis.

Example 4. ELISA

In the validation phase, baseline levels of selected proteins were determined in CSF and serum samples using commercially available ELISA kits according to the manufacturers' recommendations. CSF samples were collected as described before, and serum was obtained after centrifugation of the clotted blood and stored frozen at -

8O⁰C until used. Levels of CHBLl were measured with the METRA, ELA kit (Quidel

Corporation, San Diego, USA) in undiluted CSF and serum following a 1:2 dilution factor. Levels of ceruloplasmin were measured by quantitative competitive sandwich

ELISA (AssayPro, St Charles, USA) in diluted CSF (1:2) and serum (1:400) samples.

Levels of vitamin D binding protein were detected using a sandwich ELISA

(Immunodiagnostik AG, Germany) following CSF (1 : 100) and serum (1:40,000) dilutions. All samples were measured in duplicate. The intra-assay variability was 8.4% for CHI3L1, 5.3% for vitamin D binding protein, and 12.2% for ceruloplasmin.

The interassay variability was 15.0% for CHI3L1, 20.0% for vitamin D binding protein, and 22.7% for ceruloplasmin. Example 5. DNA analysis

The differences in the protein levels observed in CSF samples from CIS patients may be due to allelic variants present in the CHI3L1 that are disease specific. So, CIS patients that will convert to MS may have an increased frequency of particular polymorphisms of the CHI3L1 gene, compared with CIS patients who will not later convert to MS. To rule out this possibility, the present invention evaluates differences in the allelic and genotype frequencies for CHI3L1 between CIS patients that will convert to MS and patients who will remain as CIS. For this, a polymorphism located in the promoter region of the CHI3L1 gene, which has been shown to be associated with differences in the expression levels (-131C— >G), will be genotyped in both groups of patients. Genomic DNA from peripheral blood samples will be obtained using standard methods. Genotyping of the selected polymorphism will be performed by means of the 5' nuclease assay technology for allelic discrimination using fluorogenic TaqMan® probes commercially available from Applied Biosystems through the Assay-on-Demand service. Briefly, polymerase amplification will be performed in 12.5 μl reactions using 6.25 μl TaqMan® Universal PCR MasterMix, 0.625 μl of TaqMan® probe, 20 ng of genomic DNA template and 4.625 μl of MiIIiQ water. Thermal cycling and end-point PCR analysis were performed on an ABI PRISM® 7000 Sequence Detection System under specified conditions: 95°C for 10 min, and 40 cycles each of 95⁰C for 15 sec and 6O⁰C for 1 min. SNP variation was assessed by means of the allelic discrimination assay employing the Applied Biosystems software package SDS 2.1.

Example 6. Statistical analysis

Statistical analysis was performed by using the SPSS 15.0 package (SPSS Inc,

Chicago, IL) for MS-Windows. A Mann-Whitney's test was used to test for significant differences in CSF and serum levels of selected proteins between Group 1 and Group 2 CIS patients, and between CIS patients and control individuals.

Correlations between CHI3L1 levels and radiological and clinical variables in CIS patients were assessed by the Spearman rank correlation coefficient. Development of

CDMS according to baseline CSF levels of CHI3L1 was assessed by Kaplan-Meier survival analysis with Log Rank test and Cox proportional hazard regression.

Example 7. Results

Screening phase: Clinical information and CSF characteristics At baseline, demographic and clinical characteristics were comparable between CIS patients that did not convert to CDMS (Group 1) and CIS patients who converted to CDMS (Group 2) (Table 1). A higher number of CIS patients presenting with optic neuritis was observed in Group 1 compared with Group 2, although overall differences in clinical presentation were not statistically significant. As shown in Table 1, the median time between the CIS event and CSF collection was similar between Groups 1 and 2 (51.0 and 47.0 days respectively). Comparisons of CSF characteristics between groups only revealed a statistically significantly higher IgG concentration in Group 2 patients, which is associated with the IgG OB that is present in these patients.

Screening phase: Proteins associated with conversion to MS

In order to identify proteins associated with conversion to CDMS, pooled CSF samples from 30 CIS patients fulfilling Group 1 criteria and 30 CIS patients satisfying Group 2 criteria were compared using the iTRAQ proteomic technique. Only 4 proteins out of 267 identified (1.5%) showed upper regulation or down regulation in different pool comparisons: alpha- 1 antitrypsin precursor, which is a contaminant from the trypsin enzymatic digestion of proteins, and serum albumin precursor, serotransferrin precursor and plasma retinol-binding precursor, which all are common blood contaminants of CSF.

First validation phase: CSF CHI3L1 levels are increased in patients that convert to CDMS

To rule out the presence of false positive results associated with the pooling methodology and proteomic technique, 3 of the most represented differentially expressed proteins were selected for validation in individual CSF samples using an independent analytical method. CHI3L1 was selected as the unique protein identified in 5 pool comparisons. CSF and serum levels of selected proteins were determined by ELISA in a first validation cohort comprised of 36 CIS patients who remained as CIS (Group 1) and 48 CIS patients who converted to CDMS (Group 2). CSF and serum levels for these proteins were also determined in 20 individuals with other neurological diseases. As shown in Figure 1, only CHI3L1 was validated. CSF CHI3L1 levels were significantly higher in CIS patients that converted to CDMS (Group 2) compared to patients who did not convert (Group 1) (p=0.1 x 10-4) and patients with neurological disorders different to MS (Control group) (p=0.012). This finding was restricted to CSF samples, as CHI3L1 serum levels were similar between Groups 1 and 2. However, CHI3L1 serum levels were higher in CIS patients compared with controls (Group 1 vs. controls, p=0.048; Group 2 vs. controls, p=0.003).

These results point to CHI3L1 as the CSF biomarker that more reliably and best discriminates between patients that will convert to CDMS and those who will continue as CIS.

CSF CHI3L1 levels are associated with brain MRI abnormalities at baseline and disability progression during follow-up

We next investigated whether levels of CHI3L1 in CSF correlated with brain MRI- derived metrics in patients with CIS at the time of disease onset. As shown in Table 3, baseline CSF CHI3L1 levels significantly correlated with the number of gadolinium enhancing lesions and the number of T2 lesions observed in brain MRI scans performed at baseline.

Therefore, Table 3 shows the correlations between CHI3L1 CSF levels and clinical and radiological parameters at baseline and during follow-up. Data are expressed as Spearman correlation coefficient (p value). Number of patients available for each comparison is shown in parenthesis. NGD: number of gadolinium enhancing lesions. NT2L: number of T2 lesions. Statistically significant correlations are shown in bold.

Table 3

Baseline Follow-up - MRI Follow-up MRI 1 year 5 years EDSS

NGD I NT2L j NGD NT2L NGD NT2L 1 year 2 years I 3 years 4 years 5 years to

OO (N=44) i (N=44) i (N=24) <N=25) (N=I 8) (N=I 8) (N=44) (N=39) ; (N=41) CN=37) (N=29)

CHDLl : 0.32 J 0.44 j -0.23 0.07 0.12 -0.05 : 0.34 0.40 ¹ 0.47 0.38 0.29

1 ! (0.037) I (0.003) I (0.274) (0.756) (0.636) (0.833) (0.025) (0.012) J (0.002) (0.022) (0.131)

To determine whether baseline CSF CHI3L1 levels are associated with MRI abnormalities and disability progression during follow-up, analysis was restricted to those patients who will convert to MS (Group 2). As shown in Table 3, statistical significance for correlations between baseline CSF CHI3L1 levels and MRI parameters was lost at 1 and 5 year follow-up, most likely reflecting the small number of patients from whom MRI data were available. Of note, baseline CSF CHBLl levels were associated with disability progression at follow-up, as reflected by the statistically significant correlations observed between CSF CHI3L1 levels and EDSS during years 1 through 4 (Table 3).

These results indicate that CSF levels of CHI3L1 at the time of a CIS event are associated with the amount of CNS inflammation and T2 lesion burden, and more interestingly, CHI3L1 may be used as a prognostic marker for disability progression in patient who will later convert to CDMS.

Baseline high CSF levels of CHI3L1 in CIS patients are associated with shorter time to develop CDMS

Finally, in order to evaluate the association between baseline levels of CHI3L1 in CSF and time to CDMS, patients were classified into two groups (high/low) based on a cut- off value in the CSF CHI3L1 levels of 287.9 mg/ml. This cutoff value was calculated from the mean CSF CHI3L1 levels + 2 standard deviations obtained in the group of patients with other neurological disorders. Nineteen (22.6%) CIS patients with CHI3L1 levels in CSF above the cut-off value comprised the group "high", and the remaining 65 (77.4%) patients comprised the group "low". Interestingly, the time to CDMS was significantly shorter in patients with high levels of CHI3L1 in CSF at baseline compared with patients with low protein levels (Log-rank p value= 0.003) (Figure 2). The median survival times (95% confidence interval) between the CIS event and the second relapse in patients with high and low CSF CHI3L1 levels were 17.6 months (11.6 - 23.7) and 34.2 months (16.9 - 51.5) respectively. When the CSF CHI3L1 levels were incorporated into a univariate Cox regression model, the presence of high baseline CHI3L1 levels were associated with an increased risk of conversion to CDMS [hazard ratio (95% confidence interval); p-value]: 2.5 (1.3-4.7); p=0.004. These results indicate that high baseline CSF levels of CHBLl may be used as a prognostic marker for conversion to CDMS.

Determination of CSF CHBLl levels in a second validation cohort of CIS patients Finally, in a second validation phase, CSF levels of CHI3L1 were also determined by ELISA in a totally independent validation cohort comprised of 52 new CIS patients classified based on conversion to MS. Sixteen individuals with inflammatory neurological disorders were also included as control group. Similar to the findings observed in the first validation cohort, CSF CHI3L1 levels were found to be significantly increased in CIS patients who later converted to MS compared with patient who remained as CIS (p=0.018; Figure 1). Although CSF levels of CHI3L1 were also higher in patients who converted to MS compared with controls with other inflammatory neurological disorders, the difference did not reach statistical significance (p=0.351). These results reinforce the potential of CHI3L 1 to discriminate between CIS patients who convert MS and patients who remain as CIS.

Therefore, the first embodiment of the present invention refers to the use of CHI3L1 as a biomarker suitable for patients suffering from CIS, for the early diagnosis of MS or for the prognosis of MS disability progression.

The second embodiment of the present invention refers to an in vitro method for the early diagnosis of MS or for the prognosis of MS disability progression, in patients suffering from CIS, that comprises the identification of the up-regulation of the CHI3L1 gene in samples isolated from those patients suffering from CIS, wherein, in a preferred embodiment, the CHI3L1 gene expression level is evaluated by means of the CHI3L1 protein level and it is considered to be up-regulated when CHI3L1 protein level is up to the cut-off value of 287.9 mg/ml.

The third embodiment of the present invention refers to an in vitro method for screening compounds or molecules active for inhibiting the progression from CIS to

CDMS in patients suffering form CIS that have the CHI3L1 gene up-regulated, that comprises measuring the level of regulation of CHI3L1 gene in patients suffering from CIS, both in the presence and absence of said compounds or molecules, characterized in that the down-regulation of the CHI3L1 gene expression is indicative that said compounds or molecules are useful for inhibiting the progression from CIS to CDMS in patients suffering form CIS that have the CHI3L 1 gene up-regulated. The CHI3L1 protein level is considered to be down-regulated when its level is down to the cut-off value of 287.9 mg/ml.

The fourth embodiment of the present invention refers to a kit suitable for patients suffering from CIS for the early diagnosis of MS or for the prognosis of MS disability progression that comprises an antibody especially recognizing the CHI3L1 protein or probes able to hybridize with the CHI3L1 gene.

The fifth embodiment of the present invention refers to a pharmaceutical composition for inhibiting the progression from CIS to CDMS in patients suffering form CIS that have the CHI3L1 gene up-regulated, that comprises a therapeutically effective amount of an agent able to return the CHI3L1 gene expression to normal levels. The pharmaceutical comprises a therapeutically effective amount of an agent able to return the CHI3L1 gene expression to normal levels by means of the regulation of the CHI3L1 protein levels or of the transcribed mRNA levels of the CHI3L1 gene.

The last embodiment of the present invention refers to a method for inhibiting the progression from CIS to CDMS in patients suffering form CIS that have the CHI3L1 gene up-regulated that comprises the administration of an effective amount of the above mentioned pharmaceutical composition.

Therefore, all embodiments of the present invention share a single general concept based on the finding that CHI3L1 is not only up-regulated in patients with MS in general but, specifically, in patients suffering from CIS which will likely develop to CDMS. REFERENCES

1. Barkhof F, Filippi M, Miller DH, et al. Comparison of MRI criteria at first presentation to predict conversion to clinically definite multiple sclerosis. Brain. 1997;120(Pt 11):2059-2069.

2. Berger T, Rubner P, Schautzer F, Egg R, Ulmer H, Mayringer I, Dilitz E, Deisenhammer F, Reindl M. Antimyelin antibodies as a predictor of clinically definite multiple sclerosis after a first demyelinating event. N Engl J Med. 2003 JuI 10;349(2):139-45. 3. Brex PA, Ciccarelli O, O'Riordan JI, Sailer M, Thompson AJ, Miller DH. A longitudinal study of abnormalities on MRI and disability from multiple sclerosis. N Engl J Med. 2002 Jan 17;346(3): 158-64.

4. Chiasserini D, Di Filippo M, Candeliere A, Susta F, Orvietani PL, Calabresi P, Binaglia L, Sarchielli P. CSF proteome analysis in multiple sclerosis patients by two-dimensional electrophoresis. Eur J Neurol. 2008 Sep;15(9):998-1001.

5. D'Aguanno S, Barassi A, Lupisella S, d'eril GM, Del Boccio P, Pieragostino D, Pallotti F, Carelli V, Valentino ML, Liguori R, Avoni P, Bernardini S, Gambi D, Urbani A, Federici G. Differential cerebro spinal fluid proteome investigation of Leber hereditary optic neuropathy (LHON) and multiple sclerosis. J Neuroimmunol. 2008 Jan;193(l-2): 156-60.

6. Dumont D, Noben JP, Raus J, Stinissen P, Robben J. Proteomic analysis of cerebrospinal fluid from multiple sclerosis patients. Proteomics. 2004 Jul;4(7):2117-24.

7. Fisniku LK, Brex PA, Altmann DR, Miszkiel KA, Benton CE, Lanyon R, Thompson AJ, Miller DH. Disability and T2 MRI lesions: a 20-year follow-up of patients with relapse onset of multiple sclerosis. Brain. 2008 Mar;131(Pt 3):808-17.

8. Hammack, B. N., Owens, G. P., Burgoon, M. P., Gilden, D. H., Improved resolution of human cerebrospinal fluid proteins on two-dimensional gels. Mult. Scler. 2003, 9, 472-475. 9. Irani, D. N., Andersen, C, Gundry, R., Cotter, R. et al, Cleavage of cystatin C in the cerebrospinal fluid of patients with multiple sclerosis. Ann. Neurol. 2006, 26, 237-247.

10. Kuhle J, Pohl C, Mehling M, Edan G, Freedman MS, Hartung HP, Polman CH, Miller DH, Montalban X, Barkhof F, Bauer L, Dahms S, Lindberg R,

Kappos L, Sandbrink R. Lack of association between antimyelin antibodies and progression to multiple sclerosis. N Engl J Med. 2007 Jan 25;356(4):371- 8.

11. Lehmensiek V, Sϋssmuth SD, Tauscher G, Brettschneider J, FeIk S, Gillardon F, Tumani H. Cerebrospinal fluid proteome profile in multiple sclerosis. Mult

Scler. 2007 Aug;13(7):840-9.

12. Noben JP, Dumont D, Kwasnikowska N, Verhaert P, Somers V, Hupperts R, Stinissen P, Robben J. Lumbar cerebrospinal fluid proteome in multiple sclerosis: characterization by ultrafiltration, liquid chromatography, and mass spectrometry. J. Proteome Res. 2005, 5, 1647-57.

13. McDonald WI, Compston A, Edan G, Goodkin D, Hartung HP, Lublin FD, et aLRecommended diagnostic criteria for multiple sclerosis: guidelines from the International Panel on the diagnosis of multiple sclerosis. Ann Neurol 2001;50:121-7. 14. Pelayo R, Tintore M, Montalban X, Rovira A, Espejo C, Reindl M, Berger T.

Antimyelin antibodies with no progression to multiple sclerosis. N Engl J

Med. 2007 Jan 25;356(4):426-8. 15. Poser CM, Paty DW, Scheinberg L, et al. New diagnostic criteria for multiple sclerosis: guidelines for research protocols. Ann Neurol. 1983;13(3):227-231. 16. Qin Z, Qin Y, Liu S. Alteration of DBP levels in CSF of patients with MS by proteomics analysis. Cell MoI Neurobiol. 2009 Mar;29(2):203-10. 17. Recklies AD, Ling H, White C, Bernier SM. Inflammatory cytokines induce production of CHI3L1 by articular chondrocytes. J Biol Chem

2005;280:41213-21. 18. Stoop MP, Dekker LJ, Titulaer MK, Burgers PC, Sillevis Smitt PA, Luider

TM, Hintzen RQ. Multiple sclerosis-related proteins identified in cerebrospinal fluid by advanced mass spectrometry. Proteomics. 2008 Apr;8(8): 1576-85. 19. Tintore M, Rovira A, Rio J, et al. Baseline MRI predicts future attacks and disability in clinically isolated syndromes. Neurology. 2006;67(6):968-972.

20. Tintore M, Rovira A, Rio J, Tur C, Pelayo R, Nos C, Tellez N, Perkal H, Comabella M, Sastre-Garriga J, Montalban X. Do oligoclonal bands add information to MRI in first attacks of multiple sclerosis? Neurology.

2008;70(13 Pt 2): 1079-1083.

21. Van Bilsen JH, van Dongen H, Lard LR, van der Voort EI, Elferink DG, Bakker AM, et al. Functional regulatory immune responses against human cartilage glycoprotein-39 in health vs. proinflammatory responses in rheumatoid arthritis. Proc Natl Acad Sci U S A 2004; 101: 17180-5.

Claims

1. Use of CHI3L1 as a biomarker in patients suffering from CIS, for the early diagnosis of MS or for the prognosis of MS disability progression.

2. In vitro method for the early diagnosis of MS or for the prognosis of MS disability progression, in patients suffering from CIS, that comprises the analysis of the CHI3L1 gene expression level in samples isolated from those patients suffering from CIS.

3. Method, according to claim 2, wherein the samples are isolated from CSF.

4. Method, according to claim 2, wherein the CHI3L1 gene expression level is evaluated by means of the transcribed mRNA levels of the CHI3L1 gene, or its corresponding cDNA, or the CHI3L 1 protein level.

5. In vitro method, according to claim 4, wherein the CHI3L1 gene expression level is evaluated by means of the CHI3L1 protein level and said CHI3L1 gene expression is considered to be up-regulated when the level of CHI3L1 protein in samples of CSF is up to the cut-off value of 287.9 mg/ml.

6. In vitro method for screening compounds or molecules active for inhibiting the progression from CIS to CDMS, in patients suffering from CIS, that comprises measuring the level of regulation of CHI3L1 gene, both in the presence and absence of said compounds or molecules, characterized in that the down- regulation of the CHI3L1 gene expression is indicative that said compounds or molecules are useful for inhibiting the progression from CIS to CDMS, in patients suffering from CIS.

7. Method, according to claim 6, wherein the CHI3L1 gene expression level is evaluated by means of the transcribed mRNA levels of the CHI3L1 gene, or its corresponding cDNA, or the CHI3L1 protein level.

8. Method, according to claim 7, wherein the CHI3L1 gene expression level is evaluated by means of the CHI3L1 protein level and said CHI3L1 gene expression is considered to be down-regulated when the level of CHI3L 1 protein in samples of CSF is down to the cut-off value of 287.9 mg/ml.

9. Kit for the early diagnosis of MS or for the prognosis of MS disability progression, suitable for patients suffering from CIS, that comprises an antibody especially recognizing the CHI3L1 protein or probes able to hybridize with the CHI3L1 gene.

10. A pharmaceutical composition for use in the inhibition of the progression from CIS to CDMS, in patients suffering from CIS, that comprises a therapeutically effective amount of an agent able to return the CHI3L1 gene expression to normal levels.

11. Pharmaceutical composition, according to claim 10, that comprises a therapeutically effective amount of an agent able to return the CHI3L1 gene expression to normal levels by decreasing the CHI3L1 protein levels or of the transcribed mRNA levels of the CHI3L1 gene.

12. Method for inhibiting the progression from CIS to CDMS, in patients suffering from CIS, that comprises the administration of an effective amount of the pharmaceutical composition of claims 10 or 11.