WO2010014000A1

WO2010014000A1 - Susceptibility markers for multiple sclerosis

Info

Publication number: WO2010014000A1
Application number: PCT/NL2008/050531
Authority: WO
Inventors: Rogier Q. Hintzen
Original assignee: Erasmus University Medical Center Rotterdam
Priority date: 2008-08-01
Filing date: 2008-08-01
Publication date: 2010-02-04

Abstract

The present invention relates to a method for the detection of multiple sclerosis (MS) or the susceptibility for developing MS in a subject comprising detecting the presence of the single nucleotide polymorphism rsl0492972*C in a sample of a patient, and primers and probed for this method. Further, the invention relates to the diagnosis or monitoring of MS by detecting the level of expression of the gene KIF1B. Also encompassed is therapy for MS with the protein KIF1B or by gene therapy restoring the polymorphism. Further part of the invention is the use of the gene as target for drug screening.

Description

Title: Susceptibility markers for multiple sclerosis.

TECHNICAL FIELD

The present invention is in the field of disease diagnostics, including classification and prognosis of disease, in particular multiple sclerosis. The invention also relates to methods for screening candidate therapeutic compounds for use in the treatment and prevention of multiple sclerosis, and to methods of treatment and prevention of multiple sclerosis.

BACKGROUND ART

Multiple sclerosis (MS) is a complex disease resulting from genetic and environmental factors. The genetic influence on MS susceptibility is substantial, as evidenced by the 20-fold increase in risk for siblings of MS patients. Part of the high recurrence risk is explained by the MHC Class II locus (Ebers, 2008). A recent GWA study performed by the International Multiple Sclerosis Genetics Consortium (IMSGC) reported association of MS with 17 SNPs in 14 loci (Hafler et al., 2007). Although most loci were consistently associated, only variants in two genes, HLA-DRA and IL2RA, achieved genome wide significance (p-value < 5 x 10⁸).

For a third gene, IL7R, convincing functional support was obtained in several studies (Hafler et al., 2007; Lundmark et al., 2007; Gregory et al., 2007). IL2RA and IL7R are both related to T-cell function and have also been linked to other autoimmune diseases. US2006/0198822 discloses methods and compositions for the treatment and/or diagnosis of disease caused by forms of multiple sclerosis that under-express and over-express CD 127 based on the IL7R pathway. The problem with IL2RA and IL7R is, however, that these genes lack specificity for the central nervous system (CNS) pathology seen in MS patients. Hence, diagnostic methods based on IL2RA and IL7R lack specificity as do treatment methods aimed at modifying gene expression in pathways that involve IL2RA and/or IL7R.

SUMMARY OF INVENTION Technical Problem

The problem to be solved by the present invention is to provide diagnostic methods, drug screening methods and therapeutic methods that exhibit specificity for MS.

Solution to Problem

The above problem is now solved by the provision of a new diagnostic method wherein a genetic variant of the KIFlB gene is detected. In particular, a genetic variant based on the SNP rsl0492972*C was now found to be associated with a predisposition of developing MS in the general population. This SNP is located in intron 5 of KIFlB gene and may explain part of the progressive neurodegeneration seen in MS patients.

The invention thus comprises a method for the detection of multiple sclerosis (MS) or the susceptibility for developing MS in a subject comprising detecting the presence of the single nucleotide polymorphism rsl0492972*C in a sample of a patient. More specifically, the invention comprises a method for detecting a nucleotide occurrence for a single nucleotide polymorphism (SNP) indicative of multiple sclerosis or the susceptibility for multiple sclerosis, comprising: (i) incubating a sample comprising a polynucleotide with a specific binding pair member, wherein the specific binding pair member specifically binds at or near a polynucleotide suspected of being polymorphic, wherein the polynucleotide comprises one of the nucleotide occurrences corresponding to polymorphism rsl0492972*C; and ii) detecting selective binding of the specific binding pair member, wherein selective binding is indicative of the presence of the nucleotide occurrence, thereby detecting the nucleotide occurrence for the polymorphism. In a further embodiment, the invention comprises an isolated primer pair for amplifying a polynucleotide comprising a single nucleotide polymorphism (SNP) in the polynucleotide, wherein a forward primer selectively binds the polynucleotide upstream of the SNP position on one strand and a reverse primer selectively binds the polynucleotide upstream of the SNP position on a complementary strand, wherein the SNP is rsl0492972*C. More specifically an isolated primer for determining a nucleotide occurrence of a single nucleotide polymorphism (SNP) in a polynucleotide, wherein the primer selectively binds the polynucleotide upstream of the SNP position on one strand, wherein the SNP is rsl0492972*C is comprised in the invention. In another embodiment the invention comprises an isolated probe for determining a nucleotide occurrence of a single nucleotide polymorphism (SNP) in a polynucleotide, wherein the probe selectively binds to a polynucleotide comprising a particular nucleotide occurrence of a pigmentation related SNP, wherein the SNP is rsl0492972*C. In yet another embodiment the invention comprises a kit for identifying at least one single marker allele or haplotype allele of the single nucleotide polymorphism (SNP) rsl0492972*C said kit comprising an isolated primer pair according to claim 3, and optional reagents for amplifying a polynucleotide using said primer pair. Preferably said kit comprises an isolated oligonucleotide probe according to the invention, an isolated primer according to the invention, or an isolated primer pair according to the invention, or a combinations thereof, and optional reagents for amplifying a polynucleotide using said primer pair. Also part of the invention is a method for the detection of MS in a subject comprising detection of underexpression of the gene KIFlB as compared to healthy subjects and alternatively, a method for monitoring the progression of MS in a subject comprising repeated assessment of the expression of the gene KIFlB. In another embodiment the invention comprises a method for the treatment of MS comprising increasing the intracellular level of the protein KIFlB. Preferably, said method comprises administration of a pharmaceutical composition comprising the protein KIFlB. Hence, also part of the invention is the use of KIFlB as pharmaceutical, especially for MS. Also preferably, the above method comprises replacing the dysfunctional KIFlB gene of a subject harboring the SNP rsl0492972*C by a functional counterpart. Preferably the replacement only concerns the SNP rslO492972*C. In a further aspect the invention comprises a method to screen for pharmaceutical compounds comprising culturing cells harboring the SNP rsl0492972*C, adding a compound to said culture and assay for the expression of the KIFlB gene.

Advantageous Effects of Invention

First of all the invention offers a novel way to diagnose (susceptibility for) MS and a possibility to monitor the progress of the disease. This will prevent the necessity of awaiting worsening of disability in the placebo/control arm before the effectiveness of the treatment can be established. Further, it is an advantage of the present invention that it provides for a single gene that is neuronally expressed, and hence is expected to be implicated in the irreversible axonal loss and associated unremitting disability characterizing MS in the long term. Thus, the gene now found to be involved in MS is directly linked to a major characteristic of MS and accordingly provides for a method of preventing progressive disability.

BRIEF DESCRIPTION OF DRAWINGS Fig. 1 shows the association of MS with KIFlB region in Dutch genetically isolated population. Blue: directly typed SNPs; Grey: imputed

SNPs. DESCRIPTION OF EMBODIMENTS

"Polymorphisms" are allelic variants that occur in a population. The polymorphism can be a single nucleotide difference present at a locus, or can be an insertion or deletion of one or a few nucleotides. As such, a single nucleotide polymorphism ("SNP") is characterized by the presence in a population of one or two, of four nucleotides (i. e., adenosine, cytosine, guanosine or thymidine) at a particular locus in a genome such as the human genome. Accordingly, it will be recognized that, while the methods of the invention are exemplified primarily by the detection of SNPs, the detection of expression of the KIFlB gene may yield similar diagnostic potency. In any case, monitoring expression of the KIFlB gene in patients already diagnosed with MS may help in determining progression or inhibition of the progression of the disease. Haplotypes can be inferred from genotype data corresponding to certain

SNPs using the Stephens and Donnelly algorithm (Am. J. Hum. Genet. 68: 978-989,2001). Haplotype phases (i. e., the particular haplotype alleles in an individual) can also be determined using the Stephens and Donnelly algorithm. Software programs are available which perform this algorithm (e. g., The PHASE program, Department of Statistics, University of Oxford). In one example, called the Haploscope method (See U. S. Pat. Appln. No. 10/120,804) a candidate SNP combination is selected from a plurality of candidate SNP combinations for a gene associated with a genetic trait. Haplotype data associated with this candidate SNP combination are read for a plurality of individuals and grouped into a positive-responding group and a negative-responding group based on whether predetermined trait criteria for an individual are met. A statistical analysis (as discussed below) on the grouped haplotype data is performed to obtain a statistical measurement associated with the candidate SNP combination. The acts of selecting, reading, grouping, and performing are repeated as necessary to identify the candidate SNP combination having the optimal statistical measurement. In one approach, all possible SNP combinations are selected and statistically analyzed. In another approach, a directed search based on results of previous statistical analysis of SNP combinations is performed until the optimal statistical measurement is obtained. In addition, the number of SNP combinations selected and analyzed may be reduced based on a simultaneous testing procedure.

As used herein, the term "infer" or "inferring", when used in reference to a diagnosis of (susceptibility to) MS, means drawing a conclusion about the disease situation or risk of disease situation of a subject using a process of analyzing the nucleotide occurrence(s) of the SNP of the invention in a nucleic acid sample of the subject. As disclosed herein, the nucleotide occurrence (s) can be identified directly by examining nucleic acid molecules, or indirectly by examining the polypeptide encoded by the gene in which the SNP is situated, in this case the KIFlB gene.

Methods of performing such an analysis and reaching a conclusion based on that analysis are exemplified herein.

The term "primer" as used herein refers to an oligonucleotide which is capable of annealing to the amplification target nucleotide sequence allowing a DNA polymerase to attach thereby serving as a point of initiation of DNA synthesis when placed under conditions in which synthesis of primer extension product which is complementary to a nucleic acid strand is induced, i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH. The (amplification) primer is preferably single stranded for maximum efficiency in amplification. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization. The exact lengths of the primers will depend on many factors, including temperature and source of primer. A "pair of bidirectional primers" as used herein refers to one forward and one reverse primer as commonly used in the art of DNA amplification such as in PCR amplification.

The term "probe" refers to a single-stranded oligonucleotide sequence that will recognize and form a hydrogen-bonded duplex with a complementary sequence in a target nucleic acid sequence analyte or its cDNA derivative.

The terms "stringency" or "stringent hybridization conditions" refer to hybridization conditions that affect the stability of hybrids, e.g., temperature, salt concentration, pH, formamide concentration and the like. These conditions are empirically optimised to maximize specific binding and minimize nonspecific binding of primer or probe to its target nucleic acid sequence. The terms as used include reference to conditions under which a probe or primer will hybridise to its target sequence, to a detectably greater degree than other sequences (e.g. at least 2-fold over background). Stringent conditions are sequence dependent and will be different in different circumstances. Longer sequences hybridise specifically at higher temperatures. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridises to a perfectly matched probe or primer. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M Na+ ion, typically about 0.01 to 1.0 M Na+ ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes or primers (e.g. 10 to 50 nucleotides) and at least about 60⁰C for long probes or primers (e.g. greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringent conditions or "conditions of reduced stringency" include hybridization with a buffer solution of 30% formamide, 1 M NaCl, 1% SDS at 37°C and a wash in 2x SSC at 40⁰C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in O.lx SSC at 60⁰C. Hybridization procedures are well known in the art and are described in e.g. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D.,Seidman, J.G., Smith, J.A., Struhl, K. eds. (1998) Current protocols in molecular biology. V.B. Chanda, series ed. New York: John Wiley & Sons.

Methods for detecting a nucleotide change can utilize one or more oligonucleotide probes or primers, including, for example, an amplification primer pair that selectively hybridizes to a target polynucleotide, which contains one or more SNP positions. Oligonucleotide probes useful in practicing a method of the invention can include, for example, an oligonucleotide that is complementary to and spans a portion of the target polynucleotide, including the position of the SNP, wherein the presence of a specific nucleotide at the position (i. e., the SNP) is detected by the presence or absence of selective hybridization of the probe. Such a method can further include contacting the target polynucleotide and hybridized oligonucleotide with an endonuclease, and detecting the presence or absence of a cleavage product of the probe, depending on whether the nucleotide occurrence at the SNP site is complementary to the corresponding nucleotide of the probe. A pair of probes that specifically hybridize upstream and adjacent and downstream and adjacent to the site of the SNP, wherein one of the probes includes a nucleotide complementary to a nucleotide occurrence of the SNP, also can be used in an oligonucleotide ligation assay, wherein the presence or absence of a ligation product is indicative of the nucleotide occurrence at the SNP site. An oligonucleotide also can be useful as a primer, for example, for a primer extension reaction, wherein the product (or absence of a product) of the extension reaction is indicative of the nucleotide occurrence. In addition, a primer pair useful for amplifying a portion of the target polynucleotide including the SNP site can be useful, wherein the amplification product is examined to determine the nucleotide occurrence at the SNP site.

A method for inferring the presence of or susceptibility for MS in a human subject from a nucleic acid sample of the human subject by identifying a nucleotide occurrence in the sample for the SNP according to the invention can further include grouping the nucleotide occurrences of more SNPs located in the same gene or a particular area of the genome, such as the KIFlB gene, into one marker allele or more haplotype alleles. The identified single marker / haplotype alleles then can be compared to known marker / haplotype alleles such that, when the relationship of the known marker / haplotype alleles to MS is known, an inference can be drawn as to presence of or susceptibility for MS in the subject providing the nucleic acid sample. Identification of the nucleotide occurrence can be performed using any method suitable for examining the particular sample. For example, wherein the sample contains nucleic acid molecules, the identification can be performed by contacting polynucleotides in (or derived from) the sample with a specific binding pair member that selectively hybridizes to a region of the polynucleotide that includes the SNP or SNPs, under conditions wherein the binding pair member specifically binds at or near the SNP(s). The binding pair member can be any molecule that specifically binds or associates with the target polynucleotide, including, for example, an antibody or an oligonucleotide.

Methods of the invention can in principle be performed by using any nucleic acid amplification method, such as the Polymerase Chain Reaction (PCR; MuUis 1987, U.S. Pat. No. 4,683,195, 4,683,202, en 4,800,159) or by using amplification reactions such as Ligase Chain Reaction (LCR; Barany 1991, Proc. Natl. Acad. Sci. USA 88:189-193; EP Appl. No., 320,308), Self- Sustained Sequence Replication (3SR; Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), Strand Displacement Amplification (SDA; U.S. Pat. Nos. 5,270,184, en 5,455,166), Transcriptional Amplification System (TAS; Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology 6:1197), Rolling Circle Amplification (RCA; U.S. Pat. No. 5,871,921), Nucleic Acid Sequence Based Amplification (NASBA), Cleavase Fragment Length Polymorphism (U.S. Pat. No. 5,719,028), Isothermal and Chimeric Primer-initiated Amplification of Nucleic Acid (ICAN), Ramification-extension Amplification Method (RAM; U.S. Pat. Nos. 5,719,028 and 5,942,391) or other suitable methods for amplification of DNA. In order to amplify DNA with a small number of mismatches to one or more of the amplification primers, an amplification reaction may be performed under conditions of reduced stringency (e.g. a PCR amplification using an annealing temperature of 38°C, or the presence of 3.5 mM MgCb). The person skilled in the art will be able to select conditions of suitable stringency.

The primers herein are selected to be "substantially" complementary (i.e. at least 65%, more preferably at least 80% perfectly complementary) to their target regions present on the different strands of each specific sequence to be amplified. It is possible to use primer sequences containing e.g. inositol residues or ambiguous bases or even primers that contain one or more mismatches when compared to the target sequence. In general, sequences that exhibit at least 65%, more preferably at least 80% homology with the target DNA oligonucleotide sequences, are considered suitable for use in a method of the present invention. Sequence mismatches are also not critical when using low stringency hybridization conditions.

The detection of the amplification products can in principle be accomplished by any suitable method known in the art. The detection fragments may be directly stained or labelled with radioactive labels, antibodies, luminescent dyes, fluorescent dyes, or enzyme reagents. Direct DNA stains include for example intercalating dyes such as acridine orange, ethidium bromide, ethidium monoazide or Hoechst dyes.

Alternatively, the DNA fragments may be detected by incorporation of labelled dNTP bases into the synthesized DNA fragments. Detection labels which may be associated with nucleotide bases include e.g. fluorescein, cyanine dye or BrdUrd.

When using a probe-based detection system, a suitable detection procedure for use in the present invention may for example comprise an enzyme immunoassay (EIA) format (Jacobs et al., 1997, J. Clin. Microbiol. 35, 791-795). For performing detection by manner of the EIA procedure, either the forward or the reverse primer used in the amplification reaction may comprise a capturing group, such as a biotin group for immobilization of target DNA PCR amplicons on e.g. a streptavidin coated microtiter plate wells for subsequent EIA detection of target DNA -amplicons (see below). The skilled person will understand that other groups for immobilization of target DNA PCR amplicons in an EIA format may be employed.

Probes useful for the detection of the target DNA as disclosed herein will bind to at least a part of the DNA sequence region as amplified by the DNA amplification procedure that contains the SNP of the invention and will not bind to the other SNP variant(s). Those of skill in the art can prepare suitable probes for detection based on the nucleotide sequence of the SNP and its flanking sequences without undue experimentation as set out herein. Also the complementary sequences of the target DNA may suitably be used as detection probes in a method of the invention, provided that such a complementary strand is amplified in the amplification reaction employed.

Suitable detection procedures for use herein may for example comprise immobilization of the amplicons and probing the DNA sequences thereof by e.g. southern blotting. Other formats may comprise an EIA format as described above. To facilitate the detection of binding, the specific amplicon detection probes may comprise a label moiety such as a fluorophore, a chromophore, an enzyme or a radio-label, so as to facilitate monitoring of binding of the probes to the reaction product of the amplification reaction. Such labels are well- known to those skilled in the art and include, for example, fluorescein isothiocyanate (FITC), β-galactosidase, horseradish peroxidase, streptavidin, biotin, digoxigeniii, 35S or 1251. Other examples will be apparent to those skilled in the art.

Detection may also be performed by a so called reverse line blot (RLB) assay, such as for instance described by Van den Brule et al. (2002, J. Clin. Microbiol. 40, 779-787). For this purpose RLB probes are preferably synthesized with a 5'amino group for subsequent immobilization on e.g. carboxyl-coated nylon membranes. The advantage of an RLB format is the ease of the system and its speed, thus allowing for high throughput sample processing. Any suitable method for screening the nucleic acids for the presence or absence of polymorphisms is considered to be part of the instant invention. Such methods include, but are not limited to: DNA sequencing, restriction fragment length polymorphism (RFLP) analysis, amplified fragment length polymorphism (AFLP) analysis; heteroduplex analysis, single strand conformational polymorphism (SSCP) analysis, denaturing gradient gel electrophoresis (DGGE), real time PCR analysis (e.g. Taqman®), temperature gradient gel electrophoresis (TGGE), primer extension, allele-specific hybridization, and INVADER® genetic analysis assays, cleavase fragment length polymorphism (CFLP) analysis, sequence-characterized amplified region (SCAR) analysis, cleaved amplified polymorphic sequence (CAPS) analysis.

The use of nucleic acid probes for the detection of specific DNA sequences is well known in the art. Mostly these procedures comprise the hybridization of the target DNA with the probe followed by post-hybridization washings. Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl, Anal. Biochem., 138: 267-284 (1984): Tm = 81.5 ⁰C + 16.6 (log M) + 0.41 (% GC)-0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1°C for each 1 % of mismatching; thus, the hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with > 90% identity are sought, the Tm can be decreased 10⁰C. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4 °C lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10 ⁰C lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20⁰C lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45 ⁰C (aqueous solution) or 32 ⁰C (formamide solution) it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology — Hvbridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier. New York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., supra.

The development of primers and probes useful for the detection of polymorphic positions in a nucleic acid is within the realm of ordinary skill (see for instance Sambrook, J., Russell D. W., Sambrook, J. (2001) Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory Press, Plainview, N. Y.).

By using standard DNA technology it is possible to produce probes and primers that directly or indirectly hybridize to the DNA samples to be tested or cDNA produced from RNA by reverse transcription, and which can be used in assays for the detection of the SNP (s). Nucleic acid amplification techniques allow the amplification of fragments of nucleic acids, which may be present in very low amounts.

In order to develop nucleic acid-based detection methods, the SNP- specific sequences must be determined for which primers or probes may then be developed. To detect an SNP by nucleic acid amplification and/or probe hybridization, the nucleic acid may be isolated from any raw sample material, optionally reverse transcribed into cDNA and directly cloned and/or sequenced. DNA and RNA isolation kits are commercially available from for instance QIAGEN GmbH, Hilden, Germany, or Roche Diagnostics, a division of F. Hoffmann-La Roche Ltd, Basel, Switzerland.

A sample useful for practicing a method of the invention can be any biological sample of a subject that contains nucleic acid molecules, including portions of the gene sequences to be examined, or corresponding encoded polypeptides, depending on the particular method. As such, the sample can be a cell, tissue or organ sample, or can be a sample of a biological fluid such as semen, saliva, blood, and the like. A nucleic acid sample useful for practicing a method of the invention will depend, in part, on whether the SNP of the haplotype to be identified is in the coding region or in the non-coding region. In the present case, where the SNPs to be identified is in a noncoding region, the nucleic acid sample generally is a deoxyribonucleic acid (DNA) sample, particularly genomic DNA or an amplification product thereof. However, where heteronuclear ribonucleic acid (RNA), which includes unspliced mRNA precursor RNA molecules, is available, a cDNA or amplification product thereof can be used. Furthermore, while the methods of the invention generally are exemplified with respect to a nucleic acid sample, it will be recognized that particular haplotype alleles can be in coding regions of a gene and can result in polypeptides containing different amino acids at the positions corresponding to the SNPs due to non-degenerate codon changes. As such, in another aspect, the methods of the invention can be practiced using a sample containing polypeptides of the subject.

Methods of the invention can be practiced with respect to human subjects and, therefore, can be particularly useful in disease diagnostics. Using either the cloned nucleic acid as a hybridization probe, using sequence information derived from the clone, or by designing degenerative primers based on the sequence of the SNP and its flanking sequences, nucleic acid hybridization probes and/or nucleic acid amplification primers may be designed an used in a detection assay for detecting the characteristics of the one or more SNPs in a sample as defined herein.

The DNA, or alternatively, the cDNA may be PCR amplified by using for instance Pfu and Taq DNA polymerases and amplification primers specific for the SNP DNA sequences. Also complete commercially available systems may be used for PCR (e.g. available form various suppliers such as Roche Diagnostics). A suitable method may for instance include mixing into a suitable aqueous buffering system (e.g. a commercially available PCR buffer) a suitable amount of total DNA as a template (e.g. 1 to 5 μg), a suitable amount (e.g. 10 pmol) of a pair of bi-directional amplification primers, a suitable amount of dNTPs and the DNA polymerase, denaturing the nucleic acids by boiling for 1 min, and performing a cycling reaction of around 10-50 alternating cycles of stringent primer hybridization, strand elongation and denaturing, at suitable temperatures to obtain DNA copies of the DNA template as amplification product. The amount of copies produced upon a certain number of cycles correlates directly to the amount of target DNA in the DNA template. The skilled person is well aware of the available quantitative PCR methods presently available from commercial suppliers to quantify the amount of target DNA in the template. The term "hybridization signal" as used herein inter alia refers to the amount of amplification product produced upon a certain number of cycles and thus to the amount of target DNA available as template in the reaction.

In order to amplify a nucleic acid with a small number of mismatches to one or more of the amplification primers, an amplification reaction may be performed under conditions of reduced stringency (e.g. a PCR amplification using an annealing temperature of 38°C, or the presence of 3.5 mM MgC12). The person skilled in the art will be able to select conditions of suitable stringency.

The primers herein are selected to be "substantially" complementary (i.e. at least 65%, more preferably at least 80% perfectly complementary) to their target regions present on the different strands of the sequence to be amplified. It is possible to use primer sequences containing e.g. inositol residues or ambiguous bases or even primers that contain one or more mismatches when compared to the target sequence. In general, sequences that exhibit at least 65%, more preferably at least 80% homology with the target DNA or RNA oligonucleotide sequences are considered suitable for use in a method of the present invention. Sequence mismatches are also not critical when using low stringency hybridization conditions.

The detection of the amplification products can in principle be accomplished by any suitable method known in the art. The amplified fragments may be directly stained or labelled with radioactive labels, antibodies, luminescent dyes, fluorescent dyes, or enzyme reagents. Direct DNA stains include for example intercalating dyes such as acridine orange, ethidium bromide, ethidium monoazide or Hoechst dyes.

Alternatively, the DNA or RNA fragments may be detected by incorporation of labelled dNTP bases into the synthesized fragments. Detection labels which may be associated with nucleotide bases include e.g. fluorescein, cyanine dye, digoxigenin (DIG) or bromodeoxyuridine (BrdUrd).

In a quantitative PCR method, the reaction is preferably performed by using an oligonucleotide primer that contains one or more locked' nucleic acid (LNA®) monomers, or by using LNA® fluorescent probes. LNA® technology involves an oligonucleotide (probe or primer that contains one or more LNA® monomers [2'-O, 4'-C-methylene-β-D-ribofuranosyl-modified] (e.g. Petersen and Wengel, 2003. TRENDS in Biotechnology Vol.21(2): 74-81). In an LNA monomer, the ribose sugar moiety of the nucleotide is modified, while the base itself is unaltered. The result is a covalent bridge that locks' the ribose in the N-type (3'-endo) conformation, which enhances base stacking and phosphate backbone pre-organisation. This provides the oligonucleotide with improved affinity for complementary DNA or RNA sequences and therefore a higher Tm. When using LNA® primers, the detection of the double stranded amplification products may for instance be performed by using a double-stranded DNA stain, such as SYBR Green® [Molecular Probes, Inc.] (see for instance Ponchel et al. 2003, BMC Biotechnology 3:18).

Other methods of analysing the nuclei acid suitably comprise the use of a primer extension assay; a Taqman® PCR; a differential hybridization assay; an assay which detects allele-specific enzyme cleavage; and/or allele-specific PCR.

As indicated above, one of the aspects of the invention is to monitor the expression of the KIFlB gene to monitor the progression of the disease in a subject. Therefore, also the use of the KIFlB gene and its expression product, the protein KIFlB as markers for MS is encompassed in the present invention.

"Marker" and "Biomarker" are used interchangeably to refer to a gene or the polypeptide encoded by that gene that is differentially present in a samples taken from two different subjects, e.g., from a test subject or patient having (a risk of developing) MS, compared to a comparable sample taken from a control subject (e.g., a subject not having (a risk of developing) MS; a normal or healthy subject).

The phrase "differentially present" refers to differences in the quantity or frequency (incidence of occurrence) of a marker present in a sample taken from a test subject as compared to a control subject. For example, a marker can be a protein that is present at an elevated level or at a decreased level in samples from risk or diseased subjects compared to samples from control subjects. Alternatively, a marker can be a protein that is detected at a higher frequency or at a lower frequency in samples from risk or diseased subjects compared to samples from control subjects.

A protein or polypeptide is "differentially present" between two samples if the amount of the polypeptide in one sample is statistically significantly different from the amount of the polypeptide in the other sample. For example, a polypeptide is differentially present between two samples if it is present at least about 120%, at least about 130%, at least about 150%, at least about 180%, at least about 200%, at least about 300%, at least about 500%, at least about 700%, at least about 900%, or at least about 1000% greater than it is present in the other sample, or if it is detectable in one sample and not detectable in the other. "Immunoassay" is an assay that uses an antibody to specifically bind an antigen (e.g., a marker). The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein, in the present case the protein KIFlB. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). The phrase "specifically (or selectively) binds" to an antibody or specifically (or selectively) immunoreactive with", when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. The terms "affecting the expression" and "modulating the expression" of a protein or gene, as used herein, should be understood as regulating, controlling, blocking, inhibiting, stimulating, enhancing, activating, mimicking, bypassing, correcting, removing, and/or substituting said expression, in more general terms, intervening in said expression, for instance by affecting the expression of a gene encoding that protein.

Overexpression of a protein, i.e. expression that is higher than in control situations, can be achieved by providing a cell of the subject with a copy of the gene encoding the protein, by activating the endogenous promoter or by blocking inhibiting factors. The terms "subject" or "patient" are used interchangeably herein and include, but are not limited to, an organism; a mammal, including, e.g., a human and non-human primate.

As indicated above, detection of the KIFlB protein for the diagnosis of and/or monitoring of Multiple Sclerosis (MS) is one of the embodiments of the present invention. Detection may comprise the demonstration of the presence, in absolute (e.g., μg/ml) terms or in relative terms (e.g., relative intensity of signals), or of the absence of the biomarker in (a sample of) a subject. Very suitable, the amount of the biomarker relative to another protein stably present in the subject, such as a household enzyme, may be determined in order to detect the biomarker in a subject. It will be appreciated that the step of measuring the amount of a protein need not result in an exact determination of the concentration of said protein in said sample. It is sufficient that an expression of the amount is obtained relative to the amount present (or not present) in a control sample. Any (semi) quantitative method is suitable, as long as the measured amount can be compared with control or reference values.

Non-invasive methods for detecting or measuring proteins in the body of a subject (in vivo) are well known to the artisan. Such methods may include MRI, ultrasound spectroscopy, Raman spectroscopy and/or infra red spectroscopy and generally involve the use of specific labels for detection of the protein.

The steps of measuring the amount of a protein in a sample and the step of comparing the measured amount with reference values, may be performed in a single assay wherein the amount of said protein in test and control sample is determined relative to each other, for instance by using any available differential expression analysis technique. Any method suitable for analysing the differential expression of proteins between samples may be used in such instance. In order to measure the differential expression of a protein in a biological sample under an experimental condition and compare the expression with control samples or reference values, several methods may be used for labelling of the proteins. Very suitable, the proteins from the biological samples are labelled with one or more fluorescent probes (e.g. Cy3 and Cy5) using standard protein labelling protocols. Once the proteins of a biological sample (test and control) have been labelled (preferably differentially labelled using different colour probes for test and control), they can be brought in contact with a specific antibody. The binding of the antigens to the antibody may for instance be performed upon incubation of the sample with a small volume (± 50 μl) of labelled biological material. The detection of protein bound to the antibody may be based on the generation of fluorescence. Proteins that bind to the antibodies may then be detected using a fluorescent scanner and can then be analysed to determine the differential expression between the test and control sample.

In alternative procedures, the antibody assay may be used for the quantification of a protein in a biological sample using ELISA methods. The protein identified as biomarker for assessing the progression of MS as described herein may be measured more quantitatively by such procedures. To determine the concentration of a protein in a biological sample, ELISA techniques are very suitable. Such techniques involve the production of a calibration curve of the fluorescence intensity vs. protein concentration, or the use of a competitive ELISA format, wherein known amounts of unlabelled protein or antigen are provided in the test.

In yet another aspect, the present invention provides the use of a the KIFlB protein as biomarker for diagnosis of MS or for monitoring the progression of the disease. Such use involves the detection of the protein in (a sample of) the patient, and the determination whether the amount detected is above or below the reference value. As is indicated above, the presence of the SNP rsl0492972*C interacts with the expression of the KIFlB gene. A relatively low amount of protein (i.e. an amount lower than would be expected in a normal patient, i.e. a healthy patient that does not have the SNP) would then be predictive for the presence of the SNP and hence it would be predictive for the presence of MS or the susceptibility for the disease.

Further measurements of the protein levels in the same patient will then indicate whether the disease is worsening, or whether the development of MS is more imminent. If protein levels drop over time, axonal loss because of the low amount of protein is increased, resulting in an increased chance for loss of motor and sensory function in MS patients. Thus, the level of KIFlB is a good indicator for the effective of therapy against MS. Accordingly, another embodiment of the invention comprises a method for monitoring the effect of MS therapy by determining the level of KIFlB at several time points during treatment of the patient. In yet another aspect, the present invention provides a method of treating a subject having MS or being susceptible to MS, said method comprising using a biomarker as defined herein above as a therapeutic target or as a therapeutic agent. Preferably, said use of said biomarker as a therapeutic target comprises increasing the amount of KIFlB that is under- expressed in subjects having MS or being susceptible to MS.

The present invention also relates to the use of the biomarkers of the present invention as therapeutic targets. Pharmacogenetics and pharmacogenomics aim at determining the genetic determinants linked to diseases. Most of the diseases are multigenic diseases, and the identification of the genes involved therein should allow for the discovery of new targets and the development of new drugs.

Many physiological diseases are targeted by this novel pharmaceutical approach. One can name the autoimmune and inflammatory diseases, for example Addison's disease, Alopecia Areata, Ankylosing Spondylitis, Behcet's Disease, Chronic Fatigue Syndrome, Crohn's disease, Ulcerative Colitis, Inflammatory Bowel disease, Diabetes and Cardiovascular Diseases.

The biomarker of the present invention has been identified as genetic marker for predisposition or presence of the disease. The identification of this protein and its associated genetic environment provides better information of the patient and allows for the prevention or slowing of the development of the disease itself and an improved life expectancy and general health of the patient. Knowledge of the identity of genes involved in MS therefore greatly facilitates the development of prophylactic, therapeutic and diagnostic methods for this disease. Diagnosis of the genes responsible for the risk phenotype in a certain subjects allows for the design of therapies comprising the use of specific drugs, for instance, drugs directed at modulating gene expression. It is an aspect of the present invention to use the protein markers of the present invention and/or the genes encoding these proteins for the development of compounds able to alter the KIFlB gene and/or its expression products (RNA or protein), in particular in the case of under-expression of the biomarker in the subject at risk.

In a preferred embodiment of the present invention, the compound is a small molecule capable of modulating the activity or interfering with the function of the protein expression product of the gene encoding the biomarker as defined herein. The expression level of a gene can preferably be increased in a risk phenotype. The present invention therefore also provides for "enhancers", to boost the expression level of a gene encoding the biomarker associated with MS or susceptibility for MS. "Enhancers" may be any chemical or biological compound known or found to increase the expression level of genes, to improve the function of an expression product of a gene or to improve or restore the expression of a gene.

Very suitable therapies to overcome reduced expression levels of a gene or to restore the expression of a gene encoding the biomarker as disclosed herein include the replacement by gene therapy of the gene or its regulatory sequences that drive the expression of said gene. The invention therefore relates further to gene therapy, in which the dysfunctional KIFlB gene of a subject harboring the SNP rsl0492972*C is replaced by a functional counterpart, e.g. by stable integration of for instance a lentiviral vector comprising a functional gene or regulatory sequence into the genome of a subject's host cell which is a progenitor cell of the target cell-line of the subject and grafting of said transfected host cell into said subject.

Another object of the present invention is to provide a pharmaceutical composition for the treatment of patients having MS or an increased risk of developing MS comprising one or more of the "enhancers", replacement compounds, vectors or host cells according to the present invention as a pharmaceutical reagent or active ingredient. The composition can further comprise at least one pharmaceutical acceptable additive like for example a carrier, an emulsifier, or a conservative.

In addition, it is the object of the present invention to provide a method for treatment of subjects suffering from MS or an increased risk of developing MS which method comprises the administration of the pharmaceutical composition according to the invention to patients in need thereof in a therapeutically effective amount.

Small molecule pharmaceuticals

Small molecule pharmaceuticals are usually chemical entities that can be obtained by screening of already existing libraries of compounds or by designing compounds based on the structure of the targeted protein or gene. Briefly, the structure of at least a fragment of the protein is determined by either Nuclear Magnetic Resonance or X-ray crystallography. Based on this structure, a virtual screening of compounds is performed. The selected compounds are synthesized using medicinal and/or combinatorial chemistry and thereafter analyzed for their effect on the protein in vitro and in vivo. This step can be repeated until a compound is selected with the desired effect. After optimization of the compound, its toxicity profile and efficacy as therapeutic is tested in vivo using appropriate animal model systems.

Lead compounds identified are subsequently co-crystallized with the target to obtain information on how the binding of the small molecule can be improved (Zeslawska et al. 2000, J MoI Biol 301:465-75). Based on these findings, novel compounds are designed, synthesized, tested, and co- crystallized. This optimization process is repeated for several rounds leading to the development of a high-affinity compound of the invention that successfully modulates the function of its target protein. Finally, the toxicity of the compound is tested using standard assays (commercially available service via MDS Pharma Services, Montreal, Quebec, Canada) after which it is screened in an animal model system.

Pharmaceutical Compositions and Therapeutic Uses

Pharmaceutical compositions can comprise polypeptides, polynucleotides or small molecules of the claimed invention, collectively called pharmaceutical compounds herein. The pharmaceutical compositions will comprise a therapeutically effective amount of either a biomarker protein, a polynucleotides or small molecule as described herein.

The term "therapeutically effective amount" as used herein refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect. The effect can be detected by, for example, chemical markers or antigen levels. Therapeutic effects also include reduction in physical symptoms. The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by routine experimentation and is within the judgment of the clinician. Specifically, the compositions of the present invention can be used to treat, ameliorate, or prevent the occurrence of multiple sclerosis.

For purposes of the present invention, an effective dose will be from about 0.01 mg/ kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the polynucleotide or polypeptide compositions in the individual to which it is administered.

A pharmaceutical composition can also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, such as a polypeptide, polynuclotide, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.

Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. Liposomes are included within the definition of a pharmaceutically acceptable carrier.

Delivery Methods

Once formulated, the pharmaceutical compositions of the invention can be (1) administered directly to the subject; (2) delivered ex vivo, to cells derived from the subject; or (3) delivered in vitro for expression of recombinant proteins.

Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue. The compositions can also be administered into the nervous system. Other modes of administration include topical, oral, suppositories, and transdermal applications, needles, and particle guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule.

Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and described in e.g., International Publication No. WO 93/14778. Examples of cells useful in ex vivo applications include, for example, stem cells, particularly hematopoetic, lymph cells, macrophages, dendritic cells, or tumor cells.

Generally, delivery of nucleic acids for both ex vivo and in vitro applications can be accomplished by, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei, all well known in the art. Various methods are used to administer the therapeutic composition directly to a specific site in the body. Receptor-mediated targeted delivery of therapeutic compositions containing an antisense polynucleotide, subgenomic polynucleotides, or antibodies to specific tissues is also used. Receptor- mediated DNA delivery techniques are described in, for example, Findeis et al., Trends in Biotechnol. (1993) 11:202-205; Chiou et al., (1994) Gene

Therapeutics: Methods And Applications Of Direct Gene Transfer (J. A. Wolff, ed.); Wu et al., J. Biol. Chem. (1994) 269:542-46.

Pharmaceutical compositions containing polynucleotides are preferably administered in a range of about 100 ng to about 200 mg of polynucleotides for local administration in a gene therapy protocol. Concentration ranges of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of polynucleotides can also be used during a gene therapy protocol. Factors such as method of action and efficacy of transformation and expression are considerations which will affect the dosage required for ultimate efficacy of the polynucleotides. Where greater expression is desired over a larger area of tissue, larger amounts of polynucleotides or the same amounts readministered in a successive protocol of administrations, or several administrations to different adjacent or close tissue portions of, for example, a nerve ending or synaps, may be required to effect a positive therapeutic outcome. In all cases, routine experimentation in clinical trials will determine specific ranges for optimal therapeutic effect. A more complete description of gene therapy vectors, especially retroviral vectors, is contained in U.S. Ser. No. 08/869,309, which is expressly incorporated herein.

The invention will now be illustrated by way of the following, non limiting Example.

Example 1

METHODS

Patients & genotyping

All patients fulfilled either Poser's criteria for definite multiple sclerosis or McDonald's criteria for multiple sclerosis. The Netherlands MS series. In the screening phase, we performed a

GWA study on 45 MS patients from the Genetic Research in Isolated Populations (GRIP) program. As controls, we included 195 healthy individuals from the same area who were all distantly related. Details on ascertainment are given elsewhere (Hoppenbrouwers, LA. et al., 2008). The 250K Nsp array from the GeneChip Human Mapping 500K Array Set was applied to genotype with cohort genome-wide; BRMML algorithm was used for genotype calling. Details are described elsewhere (Kayser, M. et al., 2008).

For the replication cohort we recruited and ascertained MS patients as part of an ongoing nationwide study on genetic susceptibility in MS. At total of 490 MS patients were included, 370 sporadic MS patients and 120 cases from 120 multiplex MS families (i.e. parents with 2 or more affected offspring). Ten percent of the patients (n=51) had a clinically isolated syndrome at the time of enrollment. The 426 healthy controls consisted of 26 unrelated spouses, together with 400 healthy blood donors. Genotyping in the replication sample was carried out using the

MassARRAY system/Homogeneous MassExtend assay, following the protocol provided by Sequenom. PCR and extension primers were designed using the Assay Design 3.0 program (Sequenom). Briefly, 20 ng genomic DNA is PCR amplified using Titanium Taq DNA Polymerase (Clontech). PCR primers were used at 200 nM final concentrations for a PCR volume of 10 μl. The PCR condition was 95°C for 15 min, followed by denaturing at 94°C for 20 sec, annealing at 56°C for 30 sec, extension at 72°C for 1 min for 45 cycles, and finally incubation at 72°C for 3 min. PCR products were first treated with shrimp alkaline phosphatase (Sequenom) for 20 min at 37°C to remove excess dNTPs. ThermoSequenase (Sequenom) was used for the base extension reactions. Analysis and scoring were performed using the program Typer 3.3 (Sequenom).

Swedish case-control data set. In the Swedish study, 826 MS patients were collected at Karolinska University Hospital, Stockholm, Sweden. Further, 997 healthy blood donors from the Stockholm area served as controls.

Genotypes for rs 10492972 were assessed using a pre-designed TaqMan® SNP

Genotyping Assay (Assay ID:C 30400488_20, Applied Biosystems). Primers were diluted four times compared to the manufacturer's manual. Allelic discrimination was performed using the Applied Biosystems 7900HT Fast Real-Time PCR equipment and the interpretation was done using the SDS 2.2.1 software (both from Applied Biosystems).

Canadian Collaborative Project on the Genetic Susceptibility to MS (CCPGSMS). A total of 2825 individuals from 756 families were typed as part of the CCPGSMS for which the methodology has been described (Sadovnick, A.D. et al, 1998). This includes 1318 individuals with definite MS and 1507 of their unaffected first-degree relatives. The Canadian families consisted of 456 multiplex MS families and 300 parent-child trios. All genotypes were generated blind to pedigree structure and disease status of the individual. Genotyping of SNPs was performed using the Sequenom MassEXTEND protocol (www.sequenom.com). Only conservative and moderate genotyping calls were accepted in this study. Samples having aggressive or low probability quality genotypes were re-analysed. The concordance of genotyping between Affymetrix and Sequenom platforms has previously been validated (Hafler, D.A. et al., 2007).

Statistical analysis

Genome-wide association analysis was performed using GenABEL package (Aulchenko, Y.S. et al., 2007). The Armitage trend test was used to test for association the disease and genotypes (coded as 0, 1, or 2; additive model). Genomic controls was applied to correct for possible inflation of the test statistics. Furthermore, EIGENSTRAT analysis (Price, A.L. et al., 2006) with up to ten principal components was performed to ensure robustness of results against false positives.

Imputations were performed using MACH program (Li, Y. and Abecasis, G.R., 2006) with HapMap release 21 CEU population as reference. Only imputed SNPs showing the ratio of observed to the expected variance (r2) over 0.8 were considered in analysis.

In Dutch outbred population and Swedish cohorts, odds ratios and p- values were estimated using logistic regression as implemented in glm function of R v. 2.6.2. In the Canadian Collaborative Project on the Genetic Susceptibility to MS cohort, affected offspring analysis was performed with TDT as implemented in PLINK package (Purcell, S. et al., 2007). The calculations are done by permuting parent alleles while fixing the IBD status of sibs within a family. The odds ratio was calculated as described in Kazeem and Farrall (2005). For the meta-analysis was done using inverse variance pooling of log-odds ratios.

RESULTS We conducted a GWA study in 45 MS patients and 195 controls from a young genetically isolated Dutch populations using the Affymetrix 250K Nsp array from the GeneChip Human Mapping 500K Array Set. We reasoned that the relative homogeneity of the sample would be advantageous. Association analysis did not reveal any SNP achieving genome-wide significance; the lowest p-value observed being 10^~6. However, we did detect a signal in the KIFlB locus (chromosome Ip36.22), which was characterized by multiple associated SNPs (Figure 1). While the significance for individual SNPs was modest for a GWA analysis (lowest p - 0.0004 for rsl0492972), the odds ratio (OR) for rsl0492972*C was 3.12 (95% CI from 1.66 to 5.86) per allele, and 14 (52%) out of 27 SNPs in the 500 kb region surrounding rsl0492972 demonstrated association withp-values < 0.05. There is extensive linkage disequilibrium (LD) in the KIFlB locus, and the boundaries of the major haplotype block coincided well with the boundaries of association (Figure 1). When including rsl0492972 in a logistic regression analysis no other SNPs remained significant (all_/?>0.05), suggesting that association with the other 13 SNPs was most likely explained by LD with rs 10492972. Strong LD is expected to occur around a causal variant, in particular when patients are derived from a genetic isolate. Since KIFlB is known to encode a protein involved in axonal function, we followed-up rsl0492972 in three replication sets of MS patients ascertained in outbred populations: (1) a cohort from the Dutch general population (490 MS cases and 426 controls), (2) a cohort from the Swedish general population (826 MS cases and 997 controls), and (3) the Canadian Collaborative Project on the Genetic Susceptibility to MS (CCPGSMS; 1318 affected MS patients with their parents). In total, we studied 2,634 cases and 2,930 controls in the replication phase. In each replication cohort significant association of MS with the rsl0492972*C allele was demonstrated, with ORs consistently ranging from 1.3 (Swedish cohort) to 1.42 (Dutch outbred cohort). In each replication cohort, the frequency of the C allele was very similar and close to 0.34 in cases and to 0.27 in controls (Table 1).

Table 1: Results from individual studies and meta-analysis of association between rsl0492972*C (KIFlB region) and MS

Cohort No. No. RAFa ¹ RAFu 2 OR Z P cases controls

Dutch 45 195 0.43 0.21 3.12 3.55 0.0004 isolated

Dutch 490 426 0.34 0.27 1.42 3.33 0.0009 outbred

Swedish 826 997 0.34 0.29 1.30 3.66 0.0003

Canada* 1,318 1,507 0.34 0.27 1.31 3.23 0.0012

Pooled 2,679 3,125 0.34 0.27 1.34 6.23 2.5 10¹⁰

¹ RAFa: risk allele frequency in affected 2 RAFu: risk allele frequency in unaffected * Trio design, TDT analysis

A meta-analysis of the data resulted in a joint OR of 1.35 (95% CI: 1.23 — 1.48), with a significant p-value of 2.5 x 10- ¹⁰. Rsl0492972 is located in intron 5 of the KIFlB gene. The association with MS may well be explained by regulatory elements in this region. KIFlB has been linked to the inherited peripheral neuropathy Charcot-Marie Tooth (CMT2A) in a single Japanese family (Zhao, C. et al., 2001). However, later studies have shown that mutations in mitofusin-2 (MFN2), which is located 2 Mb from KIFlB, are responsible for most cases of CMT2A linked to this locus. In our study, SNPs in MFN2 and its surrounding (+/- 100 kb) did not show evidence for association to MS (all p>0.01).

Given the strong evidence for an autoimmune origin of MS, it was not unexpected that the first MS genes discovered by the IMSGC GWA (Hafler, D.A. et al., 2007) were related to T-cell function. There is however increasing evidence for neurodegenerative processes in MS pathology. Irreversible axonal loss is an important mechanism in the development of permanent neurological symptoms (Dutta, R. and Trapp, B.D., 2007; DeLuca, G.C. et al., 2006). Though primary demyelination may underlie early axonal loss, further progression of neurodegeneration occurs when the compensatory capacity of the CNS is exceeded and the threshold of axonal loss is reached. Mechanisms proposed for this loss of nerve fibers include mitochondrial dysfunction, reduced ATP production and altered axonal expression of sodium channels (Dutta, R. et al., 2006); Craner, M.J. et al., 2004). KIFlB encodes a kinesin superfamily member believed to be responsible for axonal transport of mitochondria and synaptic vesicle precursors (Boldogh, LR. and Pon, L.A., 2007). It has an ATPase binding domain and is enriched in motor neurons. Recently, dysregulation of ATP-ases and mitochondrial mislocalization has been shown to play a role in several neurodegenerative diseases (Nangaku, M. et al., 1994). KIFlB knockout mice clearly showed CNS abnormalities such as atrophy (Zhao, C. et al., 2001).

The discovery of KIFlB is based on what may be the smallest number of cases studied by a GWA study. Although the a priori power was low, even in a genetically isolated population, the ability to locate this gene is most likely due to the demonstrably extended LD around rs 1049297 in the isolate

(Service, S. et al., 2006). Genome wide significant replication of the findings in the series of MS patients from the general Dutch, Canadian and Swedish populations confirmed the association of rslO49297 to MS. Previously we have confirmed the role oiEVIδ (Hoppenbrouwers, LA. et al., 2008), one of the putative 17 MS loci proposed by the IMSGC consortium (Hafler, D.A. et al., 2007). Interestingly, both EVI5 and the newly identified KIFlB demonstrated higher ORs in our recently isolated population, as compared to the general population. This finding is likely explained by founding effects and genetic drift, having led to an enrichment of genetic variants and their effects in the genetic isolate.

Our study establishes a new genetic variant rsl0492972*C that is associated with the risk of MS in the general population. This SNP is located in intron 5 of KIFlB gene and may explain part of the progressive neurodegeneration seen in MS patients.

CONCLUSIONS

Until recently, the MHC class II locus has been the only locus consistently associated with multiple sclerosis (MS) (Ebers, G.C., 2008). Genome wide association (GWA) study has led to the confirmation of IL2R and IL7R as novel susceptibility genes involved in MS (Hafler et al., 2007). We here report a GWA study in a genetically isolated population that identified a new MS locus on chromosome 1 comprising KIFlB. This locus was confirmed in a replication set of 2,679 cases and 3,125 controls derived from four independent populations. A single nucleotide polymorphism (SNP), rs 10492972, located in intron 5 of KIFlB, was significantly and consistently associated to MS in each of the four populations (combined odds ratio for allele C 1.35, 95% confidence interval [CI] from 1.23 to 1.48, p = 2.5 x 10 ¹⁰). KIFlB is a novel, neuronally expressed, MS susceptibility gene and expectedly implicated in the irreversible axonal loss and associated unremitting disability characterizing MS in the long term (Dutta, R. and Trapp, B.D., 2007). CITATION LIST

1. Ebers, G.C. Environmental factors and multiple sclerosis. Lancet Neurol 7, 268-77 (2008).

2. Hafler, D.A. et al. Risk alleles for multiple sclerosis identified by a genomewide study. N Engl J Med 357, 851-62 (2007).

3. Dutta, R. & Trapp, B.D. Pathogenesis of axonal and neuronal damage in multiple sclerosis. Neurology 68, S22-31; discussion S43-54 (2007).

4. Lundmark, F. et al. Variation in interleukin 7 receptor alpha chain (IL7R) influences risk of multiple sclerosis. Nat Genet 39, 1108-13 (2007). 5. Gregory et al. Interleukin 7 receptor alpha chain (IL7R) shows allelic and functional association with multiple sclerosis Nat Genet. 39, 1083-91 (2007)

6. Aulchenko, Y.S. et al. Linkage disequilibrium in young genetically isolated Dutch population. Eur J Hum Genet 12, 527-34 (2004).

7. Zhao, C. et al. Charcot-Marie-Tooth disease type 2A caused by mutation in a microtubule motor KIFIBbeta. Cell 105, 587-97 (2001).

8. DeLuca, G.C, Williams, K., Evangelou, N., Ebers, G.C. & Esiri, M.M. The contribution of demyelination to axonal loss in multiple sclerosis. Brain 129, 1507-16 (2006).

9. Dutta, R. et al. Mitochondrial dysfunction as a cause of axonal degeneration in multiple sclerosis patients. Ann Neurol 59, 478-89 (2006). 10. Craner, M.J. et al. Molecular changes in neurons in multiple sclerosis: altered axonal expression of Navl.2 and Navl.6 sodium channels and Na+/Ca2+ exchanger. Proc Natl Acad

Sci U S A 101, 8168-73 (2004).

11. Waxman, S.G. Axonal dysfunction in chronic multiple sclerosis: meltdown in the membrane. Ann Neurol 63, 411-3 (2008). 12. Boldogh, LR. & Pon, L.A. Mitochondria on the move. Trends Cell Biol 17, 502-10 (2007).

13. Nangaku, M. et al. KIFlB, a novel'microtubule plus end-directed monomeric motor protein for transport of mitochondria. Cell 79, 1209-20 (1994).

14. Service, S. et al. Magnitude and distribution of linkage disequilibrium in population isolates and implications for genome-wide association studies. Nat Genet 38, 556-60 (2006). 15. Hoppenbrouwers, LA. et al. EVI5 is a risk gene for multiple sclerosis. Genes Immun (2008).

16. Kayser, M. et al. Three genome-wide association studies and a linkage analysis identify HERC2 as a human iris color gene. Am J Hum Genet 82, 411-23 (2008).

17. Sadovnick, A.D., Risch, N.J. & Ebers, G.C. Canadian collaborative project on genetic susceptibility to MS, phase 2: rationale and method. Canadian

Collaborative Study Group. Can J Neurol Sci 25, 216-21 (1998).

18. Aulchenko, Y.S., Ripke, S., Isaacs, A. & van Duijn, CM. GenABEL: an R library for genome-wide association analysis. Bioinformatics 23, 1294-6 (2007).

19. Bacanu, S.A., Devlin, B. & Roeder, K. The power of genomic control. Am J Hum Genet 66, 1933-44 (2000).

20. Price, A.L. et al. Principal components analysis corrects for stratification in genome-wide association studies. Nat Genet 38, 904-9 (2006).

21. Li, Y. & Abecasis, G.R. Rapid Haplotype Reconstruction and Missing Genotype Inference. Am J Hum Genet, 2290 (2006). 22. Purcell, S. et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet 81, 559-75 (2007).

23. Kazeem, G.R. & Farrall, M. Integrating case-control and TDT studies. Ann Hum Genet 69, 329-35 (2005).

Claims

1. Method for the detection of multiple sclerosis (MS) or the susceptibility for developing MS in a subject comprising detecting the presence of the single nucleotide polymorphism rsl0492972*C in a sample of a patient.

2. A method for detecting a nucleotide occurrence for a single nucleotide polymorphism (SNP) indicative of multiple sclerosis or the susceptibility for multiple sclerosis, comprising:

(i) incubating a sample comprising a polynucleotide with a specific binding pair member, wherein the specific binding pair member specifically binds at or near a polynucleotide suspected of being polymorphic, wherein the polynucleotide comprises one of the nucleotide occurrences corresponding to polymorphism rsl0492972*C; and ii) detecting selective binding of the specific binding pair member, wherein selective binding is indicative of the presence of the nucleotide occurrence, thereby detecting the nucleotide occurrence for the polymorphism.

3. An isolated primer pair for amplifying a polynucleotide comprising a single nucleotide polymorphism (SNP) in the polynucleotide, wherein a forward primer selectively binds the polynucleotide upstream of the SNP position on one strand and a reverse primer selectively binds the polynucleotide upstream of the SNP position on a complementary strand, wherein the SNP is rslO492972*C.

4. An isolated primer for determining a nucleotide occurrence of a single nucleotide polymorphism (SNP) in a polynucleotide, wherein the primer selectively binds the polynucleotide upstream of the SNP position on one strand, wherein the SNP is rsl0492972*C.

5. An isolated probe for determining a nucleotide occurrence of a single nucleotide polymorphism (SNP) in a polynucleotide, wherein the probe selectively binds to a polynucleotide comprising a particular nucleotide occurrence of a pigmentation related SNP, wherein the SNP is rsl0492972*C.

6. A kit for identifying at least one single marker allele or haplotype allele of the single nucleotide polymorphism (SNP) rsl0492972*C said kit comprising an isolated primer pair according to claim 3, and optional reagents for amplifying a polynucleotide using said primer pair.

7. A kit for identifying at least one single marker or haplotype allele of the single nucleotide polymorphisms (SNP) rsl0492972*C, said kit comprising an isolated oligonucleotide probe according to claim 5, an isolated primer according to claim 4, or an isolated primer pair according to claim 3, or a combinations thereof, and optional reagents for amplifying a polynucleotide using said primer pair.

8. Method for the detection of MS in a subject comprising detection of underexpression of the gene KIFlB as compared to healthy subjects.

9. Method for monitoring the progression of MS in a subject comprising repeated assessment of the expression of the gene KIFlB.

10. Method for the treatment of MS comprising increasing the intracellular level of the protein KIFlB.

11. Method according to claim 10, comprising administration of a pharmaceutical composition comprising the protein KIFlB.

12. KIFlB for use as a medicament.

13. KIFlB for use in the treatment of MS.

14. Method according to claim 10, comprising replacing the dysfunctional KIFlB gene of a subject harboring the SNP rsl0492972*C by a functional counterpart.

15. Method according to claim 14, wherein the replacement only concerns the SNP rslO492972*C.

16. Method to screen for pharmaceutical compounds comprising culturing cells harboring the SNP rsl0492972*C, adding a compound to said culture and assay for the expression of the KIFlB gene.