WO2012076922A1

WO2012076922A1 - Method of identifying a subject having or at risk of having or developing a multiple sclerosis

Info

Publication number: WO2012076922A1
Application number: PCT/IB2010/003409
Authority: WO
Inventors: Gilbert Fournie
Original assignee: Inserm (Institut National De La Sante Et De La Recherche Medicale)
Priority date: 2010-12-08
Filing date: 2010-12-08
Publication date: 2012-06-14

Abstract

The present invention relates to a method of identifying a subject having or at risk of having or developing a Multiple Sclerosis, comprising determining, in a sample obtained from said subject, the presence or absence of the VAVl haplotype comprising variant alleles in rs2546133 and rs2617822 SNPs, wherein - haplotype wherein the variant allele (C) of SNP rs2546133 and the variant allele (A) of SNP rs2617822 are present (CA haplotype) indicates an increased risk of having or being at risk of having or developing a Multiple Sclerosis, and - haplotype wherein the variant allele (C) of SNP rs2546133 and the variant allele (G) of SNP rs2617822 are present (CG haplotype) or haplotype wherein the variant allele (T) of SNP rs2546133 and the variant allele (G) of SNP rs2617822 are present (TG haplotype) indicates a decreased risk of having or being at risk of having or developing a Multiple Sclerosis.

Description

METHOD OF IDENTIFYING A SUBJECT HAVING OR AT RISK OF HAVING OR

DEVELOPING A MULTIPLE SCLEROSIS

FIELD OF THE INVENTION:

The present invention relates to a method and kits for predicting in a subject risk of having Multiple Sclerosis.

BACKGROUND OF THE INVENTION:

Multiple sclerosis (MS) is a chronic inflammatory disease with damage of myelin sheaths and nerve fibers. There are currently no preventive strategies and available therapies are either only modestly effective or are connected to rare but serious adverse events. There is solid evidence for a genetic influence in MS. Deciphering the causative genes can reveal key genetically-controlled pathways. However, the variants of genes affecting MS are likely to be numerous and each with modest effect. This is the likely reason why only a few MS genes, namely HLA class II genes, IL7R, IL2R, CLEC16A, CD58, EVI5-RPL5 have been unambiguously identified and few more are awaiting confirmation. Moreover, even less is known about the pathogenic mechanisms conveyed by MS gene variants. Identification of additional risk genes and understanding of their mechanisms of action through experimental and functional studies represent current challenges and are critical for the use of this knowledge in disease prevention or therapy.

Nonetheless, up to this point, no single biomarker is sufficiently specific to provide adequate clinical utility for the diagnosis of MS in an individual patient.

Therefore, there is a need for identifying other factors that provide a more accurate diagnosis/prognosis of MS.

The development of molecular biological techniques and the primary completion of the human genome project have enabled the detection of genetic variations that may be directly or indirectly related to a MS. Thus, the invention aims to provide a novel method for the diagnosis/prognosis of MS using a genetic factor.

Experimental autoimmune encephalomyelitis (EAE), a model for MS, has been valuable for understanding disease mechanisms and for the development of therapy. EAE has also been useful for dissecting the genetic control of neuroinflammation (Dahlman et al 2007). The relevance for human disease relies on the assumption that there are cross-species conserved mechanisms, supported by examples of disease genes defined in rodent models that also associate with human diseases e.g. CD36 and Fcgr3. The inventors previously identified one of the most significant quantitative trait locus (QTL), Eae4 on rat chromosome 9, in the (DAxBN)F2 cross with alleles from the resistant BN strain conferring less severe disease and a lower number of IFN-γ mR A-expressing cells in the CNS (Dahlman et al 2007). This QTL displays a remarkable overlap with a series of QTLs that regulate other immune-mediated diseases or associated phenotypes such as arthritis (Furaya et al 2000, Grifitths et al 2000), gold salt-triggered glomerulonephritis (Mas et al 2004), IgE response (Mas et al 2000) and CD45RC T cell subsets (Subra et al 2001, Xystrakis et al 2004), thus plausibly controlling pathways that might be central in immune regulation.

The inventors have positioned Eae4 to the Vavl gene, which codes for a guanine nucleotide-exchange factor, an intracellular signal transducer specific for hematopoietic cells (Tybulewiscz et al 2003). Variants of the human VA VI gene determined the expression of VA VI mR A and associated with MS. VA VI expression was increased in mononuclear cells from MS blood and cerebrospinal fluid and correlated with TNF and IFN-γ expression. Positioning of the Vavl gene in rat EAE4 thus defined a genetically regulated pathogenic mechanism involving regulation of proinflammatory cytokines with relevance for human inflammatory disease.

SUMMARY OF THE INVENTION:

One object of the invention is a method of identifying a subject having or at risk of having or developing a multiple sclerosis, comprising determining, in a sample obtained from said subject, the presence or absence of the VAVI haplotype comprising variant alleles in rs2546133 and rs2617822 SNPs,

wherein

- haplotype wherein the variant allele (C) of SNP rs2546133 and the variant allele (A) of SNP rs2617822 is present indicates an increased risk of having or being at risk of having or developing a Multiple Sclerosis, and

- haplotype wherein the variant allele (C) of SNP rs2546133 and the variant allele (G) of SNP rs2617822 or wherein the variant allele (T) of SNP rs2546133 and the variant allele (G) of SNP rs2617822 is present indicates a decreased risk of having or being at risk of having or developing Multiple Sclerosis.

Another object of the invention is a kit for identifying whether a subject has or is at risk of having or developing a Multiple Sclerosis, comprising

- at least one mean for detecting the SNP selected from the group consisting of rs2546133 and rs2617822 and - instructions for use

DETAILED DESCRIPTION OF THE INVENTION:

Definitions:

Throughout the specification, several terms are employed and are defined in the following paragraphs. "Risk" in the context of the present invention, relates to the probability that an event will occur over a specific time period, as in the conversion to Multiple Sclerosis, and can mean a subject's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no- conversion. Alternative continuous measures which may be assessed in the context of the present invention include time to Multiple Sclerosis conversion and therapeutic Multiple Sclerosis conversion risk reduction ratios.

"Risk evaluation," or "evaluation of risk" in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another, i.e., from a normal condition to a Multiple Sclerosis condition or to one at risk of developing a Multiple Sclerosis, or from at risk of a Multiple Sclerosis event to a more stable inflammatory disease condition. Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of Multiple Sclerosis, such as autoantibody level detection, complement components measurement, cytokine quantification, cellular population determination in serum or other fluid, either in absolute or relative terms in reference to a previously measured population. The methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion to Multiple Sclerosis, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk for a Multiple Sclerosis. In the categorical scenario, the invention can be used to discriminate between normal and other subject cohorts at higher risk for immune-mediated disease. In other embodiments, the present invention may be used so as to discriminate those having Multiple Sclerosis from normal.

"Clinical parameters or indicia" encompasses all non-sample or non-analyte biomarkers of subject health status or other characteristics, such as, without limitation, age (Age), ethnicity (RACE), gender (Sex), family history (FamHX), height (HT), weight (WT), waist (Waist) and body-mass index (BMI), as well as others such as temperature, blood pressure, respiratory and cardiac features, and cutaneous, articular, mucus or other lesions.

A "sample" in the context of the present invention is a biological sample isolated from a subject and can include, by way of example and not limitation, bodily fluids and/or tissue extracts such as homogenates or solubilized tissue obtained from a subject. Tissue extracts are obtained routinely from tissue biopsy and autopsy material. Bodily fluids useful in the present invention include blood, urine, saliva or any other bodily secretion or derivative thereof. In a preferred embodiment, the sample to be tested is blood. As used herein "blood" includes whole blood, plasma, serum, circulating epithelial cells, constituents, or any derivative of blood.

According to the invention, Peripheral Blood Mononuclear Cells (PBMCs) are the preferred cells from which VA VI polymorphisms can be detected. In a preferred embodiment, the method for diagnosis or prognosis of a Multiple Sclerosis comprises a preliminary step of extracting PBMCs.

According to the invention, the sample comprises VAV1 nucleic acid, wherein the VA VI nucleic acid is genomic DNA.

A "subject" in the context of the present invention is a human.

The term "Allele" has the meaning which is commonly known in the art, that is, an alternative form of a gene (one member of a pair) that is located at a specific position on a specific chromosome which, when translated result in functional or dysfunctional (including non-existent) gene products.

The term "polymorphism" or "allelic variant" means an alteration in the sequence of a gene. Allelic variants can be found in the exons, introns, or the coding region of the gene, or in the sequences that control expression of the gene. Complete gene sequencing often identifies numerous allelic variants (sometimes hundreds) for a given gene. The significance of allelic variants is often unclear until further study of the genotype and corresponding phenotype occurs in a sufficiently large population.

The term "Single nucleotide polymorphism" or "SNP" means a single nucleotide variation in a genetic sequence that occurs at appreciable frequency in the population. There are millions of SNPs in the human genome. Most commonly, these variations are found in the DNA between genes. When SNPs occur within a gene or in a regulatory region near a gene, they may play a direct role in disease by affecting the gene's function or expression.

The term "linkage disequilibrium" (LD) refers to a population association among alleles at two or more loci. It is a measure of co-segregation of alleles in a population. Linkage disequilibrium or allelic association is the preferential association of a particular allele or genetic marker with a specific allele, or genetic marker at a nearby chromosomal location more frequently than expected by chance for any particular allele frequency in the population.

The term "haplotype" means a 5' to 3' sequence of nucleotides found at a set of one or more polymorphic sites in a locus on a single chromosome from a single individual.

The term "VAVI " also termed "Proto-oncogene vav" or "vav 1 guanine nucleotide exchange factor" refers to a protein that in humans is encoded by the VA VI gene. The protein encoded by this proto-oncogene is a member of the Dbl family of guanine nucleotide exchange factors (GEF) for the Rho family of GTP binding proteins. VA VI gene encodes a unique protein (VAVI) with several motifs known to play a role in tyrosine mediated signal transduction, including a DBL homology (DH) domain, a pleckstrin homology (PH) domain, a Src homology 2 (SH2) domain, and two Src homology 3 (SH3) domains. The sequence of the human VA VI gene is notably available in the database under the accession number GenelD: 7409. The protein is important in hematopoiesis, playing a role in T-cell and B-cell development and activation. This particular GEF has been identified as the specific binding partner of Nef proteins from HIV-1. Coexpression and binding of these partners initiates profound morphological changes, cytoskeletal rearrangements and activation of the JNK/SAPK signalling cascade, leading to increased levels of viral transcription and replication.

The different SNPs pertaining to the invention are known per se and sequences of them are publicly available from the data base http://w\vw.ncbi.nlm.nih.gov/SNP/ or http ://hapmap .ncbi .nlm .nih. gov/.

The inventions: The present inventors have assayed for a possible statistical linkage between specific polymorphisms contained in VA VI gene from Multiple Sclerosis patient. More precisely, the present inventors have assayed for a possible statistical linkage between specific polymorphisms contained in VA VI gene from Multiple Sclerosis patient and from control population.

By genotyping nucleic acid samples originating from MS patients and control, the inventors have found that there is a high statistical linkage between (1) specific associations of SNP and haplotype markers contained in the sequence of the VA VI gene, and (2) the ability of the corresponding patients to develop Multiple Sclerosis.

As disclosed in the examples herein, the inventors have screened nucleic acid samples originating from seven well characterized cohort of patients affected with Multiple Sclerosis (MS) to assess the genomic effects of single nucleotide polymorphisms (SNP) in VA VI gene (previously reported in congress to be implicated in the rat EAE the animal model of MS but with an other haplotype)). Significant associations between Multiple Sclerosis patient and VA VI polymorphisms have been determined.

More precisely, the inventors have genotyped thirteen one single nucleotide polymorphisms (SNPs) contained in this VA VI genes, in seven large cohort of Caucasian patients with Multiple Sclerosis. For VA VI gene, SNP biallelic markers, as well as haplotypes thereof, were tested for associations between the existence of specific alleles and MS outcome.

The inventors have now identified specific SNP biallelic markers contained in the genomic sequence of the VA VI gene wherein one haplotype (CA haplotype) is more frequent in MS patient and two haplotypes (CG and TG haplotypes) are more frequent in non-MS individuals.

As explained, the VA VI gene comprises several SNPs. While studying the association between SNPs of VA VI gene polymorphism, VAVI activity and the occurrence of Multiple Sclerosis, the inventors found that specific genetic variations in the VA VI gene (SNPs rs2546133 and rs2617822) are associated with higher VA VI messenger expression and correlate with the diagnosis/prognosis of Multiple Sclerosis,.

This finding suggests a causal role of VAVI activity in Multiple Sclerosis.

The two SNPs studied in the VA VI gene are described here after: 1) HGVS Names Chromosome Transcription Ac Am rs ID 2) RefSNP Alleles: Chr position Gene Orientation Change

1) NM_005428.2:c.205- 21036T>C

rs2546133 19 6750667 VAV1 - intronic

NT_011255.14:g.6739677T>C

2) C/T

1) NM_005428.2:c.205- 19232G>A

rs2617822 19 6752471 VAV1 - intronic

NT_011255.14:g.6741481G>A

2) A/G

A first object of the invention is a method of identifying a subject having or at risk of having or developing a Multiple Sclerosis, comprising determining in a sample obtained from said subject, the presence or absence of the VA V1 haplotype comprising variant alleles in rs2546133 and rs2617822 SNPs,

wherein

- haplotype wherein the variant allele (C) of SNP rs2546133 and the variant allele (A) of SNP rs2617822 are present (CA haplotype) indicates an increased risk of having or being at risk of having or developing a Multiple Sclerosis, and

- haplotype wherein the variant allele (C) of SNP rs2546133 and the variant allele (G) of SNP rs2617822 are present (CG haplotype) or haplotype wherein the variant allele (T) of SNP rs2546133 and the variant allele (G) of SNP rs2617822 are present (TG haplotype) indicates a decreased risk of having or being at risk of having or developing a Multiple Sclerosis.

According to the invention, the presence of haplotype in which the variant allele (C) of SNP rs2546133 and the variant allele (A) of SNP rs2617822 are present indicates an increased risk of having or being at risk of having or developing an immune-mediated disease. As shown in the examples, the presence of the CA haplotype, wherein C represents the variant allele of rs2546133 and A represents the variant allele of rs2617822, is significantly associated with an increase higher VA V1 messenger expression and with an increased risk of development of Multiple Sclerosis independently of classic Multiple Sclerosis risk factors, compared to subjects that do not exhibit said haplotype. According to the invention, the presence of the haplotype in which the variant allele C of SNP rs2546133 and the variant allele G of SNP rs2617822 are present (CG haplotype) or haplotype in which the variant allele (T) of SNP rs2546133 and the variant allele (G) of SNP rs2617822 are present (TG haplotype) indicates a decreased risk of having or being at risk of having or developing a Multiple Sclerosis. As shown in the examples, the presence of the CG or TG haplotypes, wherein C represents the variant allele of SNP rs2546133 and G the variant allele of SNP rs2617822 of the CG haplotype or wherein T represents the variant allele of SNP rs2546133 and G the variant allele of SNP rs2617822 of the TG haplotype , is significantly associated to a decrease in VA VI messenger expression and is associated with a decreased risk of development of a Multiple Sclerosis, compared to subjects that do not exhibit said SNPs.

In one embodiment of the invention, the subject having or being at risk of having or developing a Multiple Sclerosis may be a substantially healthy subject, which means that the subject has not been previously diagnosed or identified as having or suffering from a an immune-mediated disease, or that has not developed a Multiple Sclerosis.

In another embodiment, said subject may also be one that is asymptomatic for the Multiple Sclerosis. As used herein, an "asymptomatic" subject refers to a subject that does not exhibit the traditional symptoms of a Multiple Sclerosis, which is diagnosed according to internationally validated criteria (McDonald et al. 2001).

In another embodiment of the invention, said subject may be one that is at risk of having or developing a Multiple Sclerosis, as defined by clinical indicia such as for example: age, gender, other biomarker (like cytokine, interleukine or ...), previous history of a Multiple Sclerosis, family history of Multiple Sclerosis, ...

In another embodiment of the invention, said subject may be one that has been previously diagnosed or identified for Multiple Sclerosis.

According to the invention, the determination of the presence or absence of said SNPs may be determined by nucleic acid sequencing, PCR analysis or any genotyping method known in the art. Examples of such methods include, but are not limited to, chemical assays such as allele specific hybridation, primer extension, allele specific oligonucleotide ligation, sequencing, enzymatic cleavage, flap endonuclease discrimination; and detection methods such as fluorescence, chemiluminescence, and mass spectrometry. For example, the presence or absence of said polymorphism may be detected in a DNA sample, preferably after amplification. For instance, the isolated DNA may be subjected to couple reverse transcription and amplification, such as reverse transcription and amplification by polymerase chain reaction (RT-PCR), using specific oligonucleotide primers that are specific for the polymorphism or that enable amplification of a region containing the polymorphism. According to a first alternative, conditions for primer annealing may be chosen to ensure specific reverse transcription (where appropriate) and amplification; so that the appearance of an amplification product be a diagnostic of the presence of the polymorphism according to the invention. Otherwise, DNA may be amplified, after which a mutated site may be detected in the amplified sequence by hybridization with a suitable probe or by direct sequencing, or any other appropriate method known in the art.

Actually numerous strategies for genotype analysis are available (Antonarakis et al., 1989; Cooper et al, 1991; Grompe, 1993). Briefly, the nucleic acid molecule may be tested for the presence or absence of a restriction site. When a base polymorphism creates or abolishes the recognition site of a restriction enzyme, this allows a simple direct PCR genotype the polymorphism. Further strategies include, but are not limited to, direct sequencing, restriction fragment length polymorphism (RFLP) analysis; hybridization with allele-specific oligonucleotides (ASO) that are short synthetic probes which hybridize only to a perfectly matched sequence under suitably stringent hybridization conditions; allele-specific PCR; PCR using mutagenic primers; ligase-PCR, HOT cleavage; denaturing gradient gel electrophoresis (DGGE), temperature denaturing gradient gel electrophoresis (TGGE), single- stranded conformational polymorphism (SSCP) and denaturing high performance liquid chromatography (Kuklin et al., 1997). Direct sequencing may be accomplished by any method, including without limitation chemical sequencing, using the Maxam-Gilbert method ; by enzymatic sequencing, using the Sanger method ; mass spectrometry sequencing ; pyro- sequencing ; sequencing using a chip-based technology and real-time quantitative PCR. Preferably, DNA from a subject is first subjected to amplification by polymerase chain reaction (PCR) using specific amplification primers. However several other methods are available, allowing DNA to be studied independently of PCR, such as the rolling circle amplification (RCA), the InvaderTMassay, or oligonucleotide ligation assay (OLA). OLA may be used for revealing base polymorphisms. According to this method, two oligonucleotides are constructed that hybridize to adjacent sequences in the target nucleic acid, with the join sited at the position of the polymorphism. DNA ligase will covalently join the two oligonucleotides only if they are perfectly hybridized to one of the allele. Therefore, useful nucleic acid molecules, in particular oligonucleotide probes or primers, according to the present invention include those which specifically hybridize the one of the allele of the polymorphism.

Oligonucleotide probes or primers may contain at least 10, 15, 20 or 30 nucleotides. Their length may be shorter than 400, 300, 200 or 100 nucleotides.

A second object of the invention is a kit for identifying whether a subject has or is at risk of having or developing an Multiple Sclerosis, comprising

- at least means for detecting the SNP selected from the group consisting of rs2546133 and rs2617822 and

- instructions for use

In one embodiment of the invention, the kit for identifying whether a subject has or is at risk of having or developing a Multiple Sclerosis, comprising:

- at least one primer and/or at least one probe for amplification of a sequence comprising a SNP selected from the group consisting of rs2546133 and rs2617822, or at least one primer and/or at least one probe for amplification of a sequence which allows the determination of the haplotype defined by the SNPs at the VA VI gene and

- instructions for use In one embodiment of the invention, the primer or probe may be labelled with a suitable marker. In another embodiment of the invention, the primer or probe may be coated on an array.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. Joint analysis of association for rs2546133-rs2617822 haplotypes with MS. (A) Association of CA haplotype in seven cohorts; over all OR: 1.18 (95% CI 1.09-

1.28); test of overall association p<2xl0-5. (B) Association of CG haplotype, over all OR: 0.86 (95% CI 0.77-0.95); test of overall association p<0.002. (C) Association of TG haplotype; over all OR: 0.90 (95% CI 0.81-1.01); test of overall association p<0.02. A fixed effect Mantel-Haenszel meta-analysis and Woolf s test for heterogeneity was performed using the meta.MH command in rmeta package in R. In all cases test of heterogeneity was non significant. The P values for over all association of these haplotypes are from an joint association test in Unphased v 3.0.13 using study cohort as a covariate (45). Figure 2. VAV1 , TNF and IFN-γ mRNA expression in MS.

(A) VAV1, (B) TNF and (C) IFN- γ mRNA expression was higher (p<0.0001) in mononuclear cells from peripheral blood of individuals with multiple sclerosis (MS)(n=417) compared to individuals with other noninflammatory neurological disorders and healthy controls (OND)(n=143). VAV1 mRNA expression levels correlated with (D) TNF (p<0.0001, r2=0.61, n=416) and (E) IFN- γ mRNA expression levels (pO.0001, r2=0.40, n=412) in mononuclear cells from peripheral blood of MS individuals. Expression was measured with quantitative real-time PCR and normalized with GAPDH. (*** corresponds to p<0.0001)

Figure 3. Association between VAV1 mRNA levels and VAV1 rs2546133-rs2617822 haplotypes.

Association of rs2546133-rs2617822 haplotypes with VAV1 mRNA expression was estimated in PBMCs of all individuals (n=551) (A) and MS patients (n=368) (B). Analysis was performed using the hap lo. score command in the Haplo Stats package in R (The R Foundation for Statistical Computing, http://www.r-project.org/foundation/) using both additive and dominant model. MS-predisposing CA haplotype (black bars) conferred higher levels of VAV1 mRNA expression (* p<0.05, ** p<0.01, *** p<0.005).

EXAMPLE: MATERIALS AND METHODS

Animals

DA.EN-Eae4 and LEW.EN-Eae4 congenic strains were developed in Sweden and France, respectively. DA and BN rats from Sweden were originally obtained from Prof. Hedrich (Hannover, Germany). BN and LEW rats from France were obtained from Centre d'Elevage Janvier (Le Genest St Isle, France). Both congenic lines were developed using the speed congenic strategy. Briefly, in each backcross generation, rats for further breeding were selected based on genotyping of eight and 23 microsatellite markers in the congenic region, from marker D9Wox24 to D9Rat20, for DA.EN-Eae4 and LEW .EN-Eae4 strains, respectively. The genetic background of rats was also screened with 100 and 220 markers, for OA.BN-Eae4 and LEW .BN-Eae4 strains, respectively. After the complete removal of the donor genome outside the congenic fragment two heterozygous rats were intercrossed to produce the homozygous congenic strain, DA.BN-Eae4(N7Fl) and LEW.BN-Eae4(N9Fl). Experimental procedures were approved by the local ethical committees.

Genotyping of animals

Primer sequences were retrieved from http://rgd.mcw.edu, except for the MJ2170 microsatellite marker and were purchased from Proligo (Paris, France). Genotypes were determined by PCR amplification of genomic DNA as previously described (Dahlman et al 1999, Mas et al 2004). Inventors predicted the most probable marker order using 794 (DAxPVG.lAVl) G10 rats.

Induction of EAE and clinical evaluation

To induce active EAE, rats were injected either with 100 mg spinal cord (WSC) homogenate with 20 mg/ml of Mycobacterium tuberculosis, strain 37 H37Ra (Difco Laboratories, Detroit, USA), 15μg recombinant MOG, in saline emulsified (1 : 1) with incomplete Freund's adjuvant (Sigma-Aldrich, St. Louis, USA) subcutaneously in the dorsal tail root or in the hind footpads with 10 μg of guinea pig MBP emulsified in IF A containing 2 mg/ml Mycobacterium tuberculosis. A total of 100 μΐ of MBP-CFA was divided equally between the rear footpads.

To induce passive EAE, immune lymph nodes from MBP-immunized donors or MBP- specific T cell lines were adoptively transferred into naive syngenic LEW recipients as previously described (42). Rats were weighed and scored daily for clinical signs of disease on a scale from 0 to 5 depending on severity: 0, normal; 1, limp tail; 2, hind limb weakness; 3, unilateral hind limb paralysis; 4, bilateral hind limb paralysis; and 5, bilateral hind limb paralysis and incontinence (death). The results are expressed either as the mean daily clinical score of each experimental group or as the median of cumulative disease score of each experimental group, calculated as the sum of the daily disease score for each individual animal.

Proliferative response and cytokines assays

Popliteal lymph node cells, collected 12 days after immunization with MBP, were stimulated with 20 μg/ml of MBP in 96-well culture plates (Costar, Cambridge, MA). Culture medium was RPMI 1640 (Gibco Life Technologies Ltd, Cergy Pontoise, France) containing 10% fetal calf serum (FCS), 1% pyruvate, 1% non essential amino acids, 1% L-glutamine, 1% penicillin-streptomycin and 2 x 10^~5 M 2-mercaptoethanol. Proliferation was assessed by [3H]thymidine incorporation during the last 18 h of a 72 h culture period. 48 hours after MBP-stimulation, supernatants were removed and IFN-γ, TNF and IL-17 were measured by luminex multiplex Biorad kits.

In another set of experiments, splenocytes from naive rats were cultured in DMEM (Gibco-BRL, Grand Island, NY) supplemented with 5% FCS, 1% L-glutamine, 1% penicillin- streptomycin and 1%) MEM (Life Technologies, Paisley, Scotland). Cells were plated at concentration of 2xl0⁶ cell/ml and either left unstimulated or stimulated with 2.5

Con A (Sigma- Aldrich, St. Louis, MO, USA) for 6h or 18h at 37°C and 5% C0₂. Levels of TNF were measured in supernatants by Elisa.

Sequencing

The sequencing templates were amplified from the genomic DNA and the sequencing reaction was carried out using the BigDye terminator v3.1 (Applied Bio systems, Inc ABI) Products were separated and recorded on ABI 3100 (Applied Biosystems, Inc ABI). Sequences were analyzed with Vector NTI software (InforMax). SNP were first detected between the DA and the BN strain in the complete coding sequence and UTRs of all genes in the region defined by Tnfsfl4 and Vavl. Identified SNPs were subsequently typed in additional rat strains using the same procedure.

Preparation of PBMCs

The MS cohort for the expression study consisted of 417 subjects (mean age 40.6 years, range 16-73 years; 65.9% females and 34.1% males; 94% with and 6% without IgG oligoclonal bands in CSF) fulfilling the McDonald criteria of MS (McDonalds et al 2001). The individuals with other neurological diseases (OND) or healthy controls (HC) consisted of 143 subjects (mean age 39.1 years, range 14-80 years; 75.5% females and 24.5% males; all without IgG oligoclonal bands in CSF). Peripheral blood was sampled in sodium citrate- containing cell preparation tubes (Vacutainer CPT, Becton Dickinson and Company, USA) and CSF in siliconized glass tubes or polypropylene tubes. CSF samples were immediately centrifuged and the pellet was recovered and stored at -80°C until use. PBMC were separated by density gradient centrifugation. More than 95% of the cells were viable. Finally, the cells were pelleted, frozen on dry ice and stored at -80°C until RNA extraction. Relative quantification of mRNAs by real-time quantitative RT-PCR

Cell pellets were lysed, total RNA was extracted (PicoPure RNA isoltion kit, Arcturus Bioscience Mt. View, CA) and DNase treated (Qiagen RNase free DNase set, Hilden, Germany). Reverse transcription was performed with 10 μΐ total RNA (5-10 ng), random hexamer primers (0.1 μg; Gibco BRL) and Superscript Reverse Transcriptase (200 U; Gibco BRL). Real-time PCR was performed using a BioRad iQ5 iCycler Detection System (BioRad Laboratories, Ltd) with a three-step PCR protocol (95°C for 10 min. followed by 50 cycles of 95°C for 15 sec, 60°C for 30 sec. and 72 °C for 30 sec.) using SYBR green fluorophore. Primers were designed with the Primer Express Software (Perkin Elmer) in our laboratory. The primer sequences for VA V1 are (forward) 5 ^' CTG CGT GGA GAA TGA GGA G 3 ^' (SEQ ID N°l) and (reverse) 5 ' CTT GTC ATA CTC TGT CAT CTT GG 3 ' (SEQ ID N°2). The primer sequences for TNF, IFN-γ and GAPDH have been reported (Khademi et al 2009). Relative quantification of mRNA levels was performed using the standard curve method, with amplification of target mRNA and GAPDH mRNA. The standard curves were created using five serial 10-fold dilutions. The relative amount of mRNA in each sample was calculated as the ratio between the target mRNA and the corresponding endogenous control GAPDH mRNA. Statistical analysis

Differences in EAE incidence and mortality were tested with Fisher's exact test. Differences in maximum score, cumulative score, onset, cytokine levels and proliferation were tested with non-parametric Mann- Whitney's U-test. To confirm significant differences the Kruskal- Wallis test with Dunn's correction was used for each rat experiment. Differences in cytokine expression between patients and controls were tested with Mann- Whitney's U-test, while the Spearman rank test was used for analysis of correlation. All comparisons were performed using GraphPad Prism version 3.0.

The probability of observing 3 crosses with a QTL and a sequence difference and 5 crosses without QTL and sequence difference for the R63W SNP in Vavl exon 1 was estimated using the binominal distribution, estimating the expected proportion in sequence difference as that observed in all theoretically possible crosses of the strains.

Clinical material for association studies The first Swedish multiple sclerosis case-control study consisted of 1039 subjects originating from Sweden or other Nordic countries. The patients fulfilled the McDonald criteria for definite MS (McDonalds et al 2001) and were recruited by neurologists at the Karolinska University Hospital in Stockholm. There were two control cohorts the first sample consisted of 752 blood donors and the second cohort consisted of 454 female blood donors. These subjects lived in the Stockholm area and originated from Sweden or other Nordic countries.

To follow-up our original association with MS was investigated in an additional 2546 patients and 3943 controls from Sweden, Norway, Denmark, Finland and 1633 families from Canada and France. Oral and/or written informed consent was given from all individuals involved in the study. The local Ethical Committees in Sweden, Norway Denmark, Finnland, France and Canada have approved the study.

Haplotype tagging

Genotypes available at the HapMap (http://www.hapmap.org/index.html.en) from

Caucasian families for SNPs covering the TRIP 10, SH2D3A and VA V1 genes were used for identifying tagging SNPs (tSNPs). 113 SNPs from rs340139 to rs461970 were used to identify tSNPs the region. We used TAGGER in Haploview v3.32 (http://www.broad.mit.edu/mpg/haploview/index.php) and ENTROPY as implemented in marker Marker (http://www.gmap.net/perl/marker/marker_entry) for identifying tSNPs in an unstructured way not limited by recombination block structure which resulted in 40 tSNPs.

Genotyping of human material

SNPs were genotyped using the TaqMan method (Applied Biosystems, Inc ABI). Of the 40 selected assays 2 could not be designed and 7 SNPs were either not polymorphic or had assays that did not result in clear distinction between the alleles. The remaining 31 SNPs were analyzed in all MS patients in the first Swedish cohort and the first set of blood donor controls. It should be noted that these 31 SNPs do not extract all the common haplotypes in this genetic region. Four SNPs (rs2546133, rs308197, rs308194 and rs331679) were genotypes twice in the first MS cohort. The concordance between the two runs was 98.2%. All discordant genotyped were checked, if the quality differed the best was kept, if it was not obvious that one was better than the other both were removed from the analysis. All markers were tested for Hardy- Weinberg equilibrium, one marker (rs339404) showed an increase and one a decrease (rs2660485) in observed homozygotes compared to expected among controls (p<0.02 and <0.05 respectively). All other markers were in Hardy- Weinberg equilibrium. Eight markers (rs2617818, rs2546133, rs2617822, rs2617823, rsl64022, rs3786688, rs308194 and rs331679) were genotyped in the second set of blood donor controls. An additional 18 SNPs were added in regions which in the initial association analysis showed evidence of association or which had been reported to be coding SNPs. These were genotyped in both the Swedish cohort la and lb.

Association analysis

The initial association analysis was performed by testing for difference in allele frequency between patients and controls using single maker analysis in Haploview 3.32. This was followed by an association analysis for haplotypes with in blocks of high linkage disequilibrium. A permutation test (n=10 000) as implemented in Haploview was employed to correct for multiple comparison including both individual markers and haplotypes. The evidence of association was also estimated using the Unphased program v3.0.13 and PLINK with similar results. The results found with Haploview are presented in the text unless otherwise stated. Unphased v3.0.13 was used to perform a joint test of association for all the MS cohorts, the study cohort was used as a covariate. Association analysis between VAV1 mRNA levels and haplotype was performed in the Haplo Stats package (v 1.2.1) in R using the haplo. score command.

RESULTS

Refinement of Eae4 to a ~1 cM region with a panel of interval-specific congenic lines

To identify the gene underlying Eae4, DA.BN-Eae4 and LEW.BN-Eae4 strains

(henceforth denoted DA.BN and LEW.BN) were created by introgression of a region encompassing Eae4 from the resistant BN strain into the susceptible DA and LEW backgrounds, respectively. Alleles from the resistant BN strain at Eae4 conferred less severe disease induced with whole spinal cord (WSC), myelin oligodendrocyte glycoprotein (MOG) or myelin basic protein (MBP). In chronic disease models, WSC- and MOG-induced EAE in DA rats, BN alleles at Eae4 conferred better recovery from the initial bout with significantly lower cumulative clinical score (p<0.03 and 0.05, respectively) and in two out of four experiments there was lower mortality (p<0.001) compared to the susceptible DA strain. In the monophasic MBP-induced EAE in LEW rats, BN alleles at Eae4 conferred significantly lower incidence (p<0.05) and milder disease (p<0.008) compared to the susceptible LEW alleles.

To narrow the Eae4 interval, we created interval-specific congenic lines. In the DA/BN strain combination, the R25 congenic line displayed significantly less severe EAE (p<0.05), compared to the susceptible background DA strain whereas R15 did not differ from DA, thus narrowing Eae4 to a ~2 cM region. In the LEW/BN strain combination, the Ra congenic line displayed similar disease severity as parental LEW strain, narrowing Eae4 to ~1 cM region fully included in the 2 cM region defined in the DA/BN combination. Taken together, these data showed that susceptibility to EAE is regulated by gene(s) located in a lcM interval between D9Wox24 and D9Got8 markers.

Impact of Eae4 on encephalogenicity and cytokine profile of MBP-specific CD4 T cells

We performed adoptive transfer experiments to investigate if genes within Eae4 had impact on the encephalogenicity of donor derived T cells or on the susceptibility of target organ to neuroinflammation. Classical EAE developed in all naive LEW rats given 25x10⁶ MBP-stimulated lymph node cells from LEW or Ra rats immunized with MBP. By contrast, LEW rats receiving MBP-stimulated cells from LEW.BN rats developed a mild EAE (p<0.05;). Similar results were obtained when LEW.BN rats were used as recipients. This reduced ability of lymph node cells from LEW.BN resistant rats to transfer EAE is a stable phenomenon that is persistent despite repeated in vitro stimulation with the autoantigen and APC from syngeneic naive rats. Indeed, MBP-specific T cell lines derived from LEW.BN rats, after 3 rounds (p<0.03;) or 4 rounds (p<0.006) of stimulation, have a reduced capacity to transfer EAE as compared to those obtained from LEW rats. According to the well- established role of inflammatory cytokines in EAE development, we investigated the influence of Eae4 on cytokine production by myelin-specific T cells. In vitro, upon stimulation with MBP, immune lymph node cells from MBP-immunized LEW.BN rats proliferated less (p<0.01;) and produced significantly lower amounts of TNF (p<0.006;) and IFN-γ (p<0.01;) than immune lymph node cells from MBP-immunized Ra rats. The production of IL-17 was similar in both groups. Together, these experiments demonstrated that the Eae4 locus contains gene(s) that regulate proinflammatory cytokine production and encephalogenicity of myelin specific T cells, thus providing a downstream mechanism of action of Eae4 gene(s). A SNP-phenotype analysis across rat strains positioned Vavl as the gene underlying Eae4

The analysis of TNF secretion by naive splenocytes stimulated with Con A showed that the regulation of TNF expression co-segregated with the regulation of EAE severity in all screened interval-specific congenic lines. Recently we made a congenic line with a 120 Kb region from the susceptible LEW strain on the resistant BN background comprising five genes, extending from tumor necrosis factor ligand superfamily member 14 (Tnfsfl4) gene to the Vavl gene. The EAE-resistant BN background precluded testing of the influence of the region on EAE. However, investigation of TNF production confirmed that this 120 Kb region regulated TNF levels (p<0.009). The inventors therefore focused on the five genes contained in this region, tumor necrosis factor ligand superfamily member 14 (Tnfsfl4), complement 3 precursor (CJ), G protein coupled receptor 108 (Gprl08), Cdc42-interacting protein 4 (Cip4/Tripl0) and Vavl oncogene (Vavl) (the homologous region is available at http://www.ensembl.org/, MMU17: 57.3-57.45 Mb). Since inventors did not find any differences in mRNA expression of these five genes between the parental strains, they sequenced their coding regions in the DA and BN strains. The identified SNPs were then genotyped in six additional rat strains selected according to available information on linkage analysis for different inflammatory diseases. Assuming that the same gene may be responsible for susceptibility in different inflammatory models, the SNPs should be shared between strains in which there was no evidence for linkage and should differ between strains in which there was evidence for linkage. The SNP in exon 1 (CGG→ TGG) resulting in an Arginine to

Tryptophan (R63W) substitution in the conserved calponin homology (CH) domain of Vavl is the only polymorphism that fulfills these criteria (p<0.005). It segregates between strains in which there was evidence for linkage to inflammatory diseases, such as DA/BN, LEW/BN and BB/BN (Dahlman et al 1999, Furuya et al 2000, Griffiths et al 2000, Mas et al 2004, Mas et al 2000, Subra et al 2001) (present study) and it is shared among all other strains in which there was no evidence for linkage. The BB/BN cross used to identify the Cial5 locus that controls collagen induced arthritis (Furuya et al 2000) is especially informative as Vavl is the only gene in the Eae4 region that differs between BB and BN strains.

Since the BN alleles at Eae4 on the susceptible DA/LEW strain background were sufficient to result in the same low TNF levels as in the donor BN strain the inventors used this strong association between SNP(s) in the Eae4 region and this phenotype to identify the polymorphism in five genes that correlated with TNF levels. Again, the polymorphism causing R63W substitution in Vavl was the only SNP that correlated with TNF levels, with R63 predisposing for high TNF expression and W63 predisposing for low TNF expression. With the positioning of Vavl variants being instrumental for inflammatory experimental disease, we decided to investigate whether VA VI is associated with human multiple sclerosis.

Association of VA V1 with MS

The inventors first investigated the syntenic human 19pl3.3 region comprising VA V1 by testing association of 47 tagging SNPs in a case-control study consisting of 1039 MS patients and 1206 controls from Sweden. Inventors observed evidence for association for several SNPs and haplotypes in VA V1 gene, most apparent in the block 6.

Markers in the block 6 haplotype were analyzed in an additional 2546 patients and 3943 controls from Sweden, Norway, Denmark, Finland and 1648 families from Canada and France (amounting to 12 735 individuals). The inventors tested association to haplotypes formed by rs2546133 and rs2617822. The common CA haplotype (-85%) had a higher frequency among cases or was over-transmitted to cases in six-out-of-seven cohorts and a joint analysis of all cohorts resulted in p<2xl0-5, OR =1.18 (Table 1 and Figure 1A). Both the CG and TG haplotypes were less frequent among patients compared to controls and showed a decreased transmission to patients with OR 0.86 p<0.002 and 0.90 p<0.02, respectively (Table 1 and Figure 1B,C).

To estimate the probability that our report of association between MS and VAV1 is false we calculated the false positive report probability (FPRP) according to the three factors that determine its magnitude, namely (i) the prior probability of a true association of the tested genetic variants with the disease (π), (ii) the observed P value and (iii) the statistical power to detect the OR of the alternative hypothesis at the given P value. Given a π value in the range 0.01-0.002, a conservative estimate for our data, we found a FPRP below 10%, strongly arguing that our reported association is true. Inventors therefore conclude that there is an association of VA Vl rs2546133-rs2617822 haplotypes in intron 1 with human MS.

VAV1, TNF and IFN-γ expression in MS

The inventors then investigated expression of VAV1 mRNA and correlation to TNF and IFN-γ expression in individuals with MS. VAV1 mRNA was significantly (p<0.0001) up- regulated in mononuclear cells of MS patients compared to controls in peripheral blood (Figure 2A) and cerebrospinal fluid (CSF) (not shown). Up-regulation of TNF and IFN-γ was also significant (p<0.0001) in MS patients compared to controls in blood (Figure 2B,C) and CSF (not shown). Moreover, levels of VAVl and TNF (r2=0.61, p<0.0001), and VAVl and IFN-γ (r2=0.40, p<0.0001) were correlated both in MS peripheral blood cells (Figure 2D,E) and in CSF (not shown).

The inventors then demonstrated that the CA haplotype in VAVl intron 1, which associated to an increased risk for MS, also associated with an increased VAVl expression in PBMC as analyzed in cases and controls (p<0.0008 in dominant model) (Figure 3A) and in MS patients separately (p<0.03 and p<0.0003 in additive and dominant model, respectively) (Figure 3B). Together these data provide additional support for VAVl being an important player in MS pathogenesis.

Table 1. Association of haplotype formed between rs2546133 and rs2617822 with multiple sclerosis

Haplotype estimation and association analysis was carried out using Unphased v 3.0.13 (45). For all but the French and Canadian MS the p-value corresponds to a test of the hypothesis of equal minor allele frequencies among patients and controls. For the French and Canadian MS study the p-value corresponds to a test of distortion of transmission to affected offspring away from expected 50% transmission. The over all test of association with MS was carried out using a joint association test in using study cohort as a covariate.

CG TQ

Patients

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Swiiiffli tS 1148 ai «27 84.8 o.i Si 63 Κβ 7.7 0,45 S3 7 ¾ (4

917 «6.7 S74 S3.4 72 i.S ϊ59 0.i 67 i.3 75 7.2 0.43 anish SOI S5.S a57 57 S.S 75 e.« o.ss 84 8,5 32 ?,¾ as

F:titifei-!⁺ (444 §4,8 S .i 057 47 3..1 S3 S ¾? 35 2.2 45 2.4 0,69

9 3 5)3. a SS28 495 031 103 SO.5 Iff! 43.5 i3.92 J06 45.7 126 54.3 0.)S

Ca:>i !i.¾f}" 1433 51.S 1 133 48,4 ! i« 4>5.2 162 S3.iS a.tfi 148 4S 154 51 0.12

Ovetel MS SsSJ ¾S H 2S 85-t ο.οοοκ 658 :: > SS 7,4 0,002 65 6.4 950 7.0 0.02

^A Sweden I contains Sweden I and all Swedish RA controls.

^B Genotypes for rs2546133 were imputed using the IMPUTE program and haplotype structure from HapMap. ^c Transmitted and untransmitted haplotypes to affected individuals. DISCUSSION

The inventors identified Vavl as a major gene in Eae4 locus. In the rat, Vavl variants are associated with the control of neuro inflammation and lymphocyte activation. Coherent with the rat data, human VA VI variants are associated with MS susceptibility and determined VAVl expression. A central role of VAVl is furthermore supported by its up-regulation in MS and by its correlation with TNF and IFN-γ expression in MS peripheral blood cells and in CSF. Thus, our data illustrate the power of defining a genetically controlled pathogenic mechanism using a rat model mimicking a complex human inflammatory disease.

VAVl is a cytoplasmic guanine nucleotide exchange factor (GEFs) for Rho-family

GTPases, known to play an important role in development and activation of T and B cells (Tybulewicz et al 2003). Activation of VAVl leads to induction of Ca2+-flux and activation of NF-κΒ, NFAT and ER -MAP pathways. However, besides T- and B-cell receptors, activation of VAVl occurs downstream of many receptors including FcsRI, FCYRI/II/III, growth factor receptors, integrins, cytokine and chemokine receptors. A potential role of VAVl in EAE has been indicated by the resistance of Vavl knock-out mice to EAE induction (Korn et al 2003). Nevertheless, it is difficult to draw conclusions from Vavl gene deletion experiments since this leads to reduced numbers of T and B cells and severely impairs their function (Zhang et al 1995). The reported disease protection is likely related to the general dysfunction of lymphocytes, not necessarily implicating Vavl as the susceptibility gene. Thus the consequences of such complete gene deletion are very different from the demonstration herein that the naturally occurring genetic variants of Vavl affect the outcome of inflammation and lymphocyte activation.

Having positioned the regulation of rat inflammatory disease to Vavl we studied the human ortholog. Combined analysis of 7 independent cohorts including 12 735 individuals showed a positive association with MS for the CA haplotype (p<2xl0-5, odds ratio 1.18) and negative association for the CG (p<0.002, odds ratio 0.86) and TG haplotypes (p<0.02, odds ratio 0.90). The odds ratios conferred by these haplotypes are in the range that is expected for non-HLA genes, as demonstrated for IL7R and IL2R (Hafler et al 2007). Furthermore, the levels of significance obtained in the size of the material used in our study conforms well to theoretical calculations of expected p-values at these particular odds ratios (Sawcer 2008). FPRP calculation as indicated in the results strongly supports the validity of the reported association. A role of VAVl in MS is further supported by higher mRNA levels in PBMC and CSF of MS patients and by correlation between VAVl and proinflammatory cytokine expression. Moreover, higher levels of VAVl are conferred by the MS-predisposing VAVl haplotype, with p<3xl0-4 in 368 MS patients. Further support for VAVl being instrumental in inflammatory disease derives from modest associations with Type 1 diabetes and Crohn^'s disease in the recent Wellcome Trust GWAS. Interestingly, Cbl-b which is a negative regulator of Vavl has been implicated in type 1 diabetes. The lack of association in the MS GWAS screen (Hafler et al 2007) can be explained by the gap of 25 kb between the markers included in VAVl intron 1 in that GWAS. The linkage disequilibrium between the markers included in this screen and those that we find associated in this report was low (r2<0.10). The evidence for a role of VAVl in several inflammatory diseases in humans is consistent with the overlap of Eae4 with QTLs that regulate many inflammatory phenotypes and diseases in rodents (Dahlman et al 1999, Furuya et al 2000, Griffiths et al 2000, Mas et al 2004, Mas et al 2000, Subra et al 2001, Xystrakis et al 2004). Hence VAVl contributes to susceptibility of MS and also potentially to other chronic inflammatory diseases.

The exact molecular mechanisms underlying the effects of VAVl gene variants in both rats and humans remain to be established. A straightforward interpretation of our findings is that the disease-predisposing variant of Vavl allows a more potent activation of autoaggressive lymphocytes producing proinflammatory cytokines which promote a more severe clinical disease and relapses. The observed strong effect of Vavl on TNF and IFN-γ is very interesting in view of the overall important role of these cytokine on CNS inflammation. TNF was initially thought to promote EAE and demyelination, while later studies revealed down-regulation of CNS inflammation. IFN-γ has a similarly complex role in EAE and possibly also in MS. A disease-promoting role has been suggested by increased levels of this cytokine in MS and by myelin antigen specific T cells, and worsening of MS by treatment with the cytokine. Paradoxically, experiments with IFN-γ treatment, neutralization and IFN-γ knock-out mice suggest a disease down-regulatory role. Surprisingly, IL-17, argued to be the prime disease-mediating cytokine in EAE is similarly produced by MBP specific T cells independently to their encephalogenicity in our experiments. Thus, our findings put into focus the regulation Thl-mediated tissue damage by VAVl as a mechanism of susceptibility to CNS inflammation. REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS:

1. A method of identifying a subject having or at risk of having or developing a Multiple Sclerosis, comprising determining in a sample obtained from said subject, the presence or absence of the VAVl haplotype comprising variant alleles in rs2546133 and rs2617822

SNPs,

wherein

2. The method according to any one of claim 1, wherein the sample consists in blood, or a preparation of Peripheral Blood Mononuclear Cells (PBMCs).

3. The method according to any one of claim 1 and 2, wherein the presence or absence of said SNP is determined by nucleic acid sequencing or by PCR analysis.

4. A kit for identifying whether a subject has or is at risk of having or developing a Multiple Sclerosis, comprising :

- at least means for detecting the SNPs selected from the group consisting of rs2546133 and rs2617822 and

- instructions for us.

5. A kit according to claim 4, comprising:

- at least one primer and/or at least one probe for amplification of a sequence comprising a

SNP consisting of rs2546133 and rs2617822, or at least one primer and/or at least one probe for amplification of a sequence which allows the determination of the haplotype defined by the SNPs at the VA VI gene and

- instructions for use.