EP1863927A2 - Compositions and methods for treating and diagnosing inflammatory disorders - Google Patents

Compositions and methods for treating and diagnosing inflammatory disorders

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Publication number
EP1863927A2
EP1863927A2 EP06725045A EP06725045A EP1863927A2 EP 1863927 A2 EP1863927 A2 EP 1863927A2 EP 06725045 A EP06725045 A EP 06725045A EP 06725045 A EP06725045 A EP 06725045A EP 1863927 A2 EP1863927 A2 EP 1863927A2
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EP
European Patent Office
Prior art keywords
seq
nrgl
polypeptide
psoriasis
subject
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP06725045A
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German (de)
French (fr)
Inventor
Ilya Chumakov
Daniel Cohen
Fabio Macciardi
Benedicte Belloir
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Ares Trading SA
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Ares Trading SA
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Priority to EP06725045A priority Critical patent/EP1863927A2/en
Publication of EP1863927A2 publication Critical patent/EP1863927A2/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates, generally, to methods and compositions for detecting or treating inflammatory disorders.
  • the inflammatory disorder may be an inflammatory CNS disorder such as multiple sclerosis (MS), an inflammatory disorder involving multiple organ systems as in the case of systemic lupus erythematosus (SLE) or an inflammatory skin disorder such as psoriasis.
  • MS multiple sclerosis
  • SLE systemic lupus erythematosus
  • psoriasis inflammatory skin disorder
  • the present invention more particularly relates to the human NRGl gene, which can be used for the diagnosis, prevention and treatment of multiple sclerosis, systemic lupus erythematosus, psoriasis and related disorders, as well as for the screening of therapeutically active drugs.
  • the invention further discloses specific polymorphisms, splice variants or alleles of the NRGl gene that are related to inflammatory disorders, such as multiple sclerosis, systemic lupus erythematosus or psoriasis, as well as diagnostic tools and kits based on these susceptibility alterations.
  • the invention can be used in the diagnosis or detection of the presence, risk or predisposition to, as well as in the prevention and/or treatment of multiple sclerosis, systemic lupus erythematosus, psoriasis and related disorders.
  • Psoriasis is considered mild if it affects less than 5% of the surface of the body, moderate if 5-30% of the skin is involved, and severe if the disease affects more than 30% of the body surface.
  • the invention can be used in the diagnosis of predisposition to, detection, prevention and/or treatment of multiple sclerosis, systemic lupus erythematosus, psoriasis and related disorders in any mammalian subjects, particularly human patients.
  • the invention relates to antibodies, which specifically bind to the NRGl polypeptide.
  • multi sclerosis may be defined as in the DSM-IV classification (Diagnosis and Statistical Manual of Inflammatory CNS Disorders, Fourth Edition, American Psychiatric Association, Washington D.C., 1994).
  • systemic lupus erythematosus may be defined as by Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF et al. "The 1982 revised criteria for the classification of Systemic lupus erythematosus” Arthritis Rheum 1982;25: 1271 -1277.
  • Duchenne muscular dystrophy is a fatal disorder caused by absence of dystrophin.
  • Krag et al. PNAS, September 2004; 101(38):13856-60
  • NRGl isoform 6 tested the ability of NRGl isoform 6 to improve the dystrophic phenotype in the mdx mouse model of DMD.
  • Intraperitoneal injections of the small peptide encoding the epidermal growth factor-like region of heregulin ectodomain for 3 months in vivo resulted in upregulation of utrophin, a marked improvement in the mechanical properties of muscle as evidenced by resistance to eccentric contraction-mediated damage, and a reduction of muscle pathology.
  • gene as used herein shall be construed to include any type of coding nucleic acid region, including genomic DNA (gDNA), complementary DNA (cDNA), synthetic or semi- synthetic DNA, any form of corresponding RNA (e.g., mRNA), etc., as well as non coding sequences, such as introns, 5'- or 3 '-untranslated sequences or regulatory sequences (e.g., promoter or enhancer), etc.
  • the term gene particularly includes recombinant nucleic acids, i.e., any non naturally occurring nucleic acid molecule created artificially, e.g., by assembling, cutting, ligating or amplifying sequences.
  • a gene is typically double-stranded, although other forms may be contemplated, such as single-stranded. Genes may be obtained from various sources and according to various techniques known in the art, such as by screening DNA libraries or by amplification from various natural sources. Recombinant nucleic acids may be prepared by conventional techniques, including chemical synthesis, genetic engineering, enzymatic techniques, or a combination thereof. The term “gene” may comprise any and all splicing variants of said gene.
  • the present invention also encompasses fragments of the NRGl gene.
  • a fragment of a gene designates any portion of at least about 8 consecutive nucleotides of a sequence of said gene, preferably at least about 15, more preferably at least about 25 nucleotides, further preferably of at least 35, 50, 75, 100, 150, 200 or 300 nucleotides. Fragments include more particularly all possible nucleotide length between 8 and 500 nucleotides, preferably between 15 and 300, more preferably between 25 and 200.
  • the present invention also encompasses DNA sequences, which have at least 40% identity with the NRGl sequence. More preferably, they have at least 50%, at least 60%, at least 70%, at least 80% or, most preferably, at least 90% identity thereto.
  • Identity reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing the sequences. In general, identity refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of the two polynucleotides or two polypeptide sequences, respectively, over the length of the sequences being compared.
  • the present invention also encompasses DNA sequences, which hybridize to the complement of the NRGl nucleic acid sequence under moderately stringent conditions or under highly stringent conditions.
  • stringent conditions refers to hybridization and subsequent washing conditions, which those of ordinary skill in the art conventionally refer to as “stringent”. See Ausubel et ah, Current Protocols in Molecular Biology, supra, Interscience, N.Y., ⁇ 6.3 and 6.4 (1987, 1992), and Sambrook et a (Sambrook, J. C, Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  • examples of stringent conditions include washing conditions 12-20°C below the calculated Tm of the hybrid under study in, e.g., 2 x SSC and 0.5% SDS for 5 minutes, 2 x SSC and 0.1% SDS for 15 minutes; 0.1 x SSC and 0.5% SDS at 37°C for 30-60 minutes and then, a 0.1 x SSC and 0.5% SDS at 68°C for 30-60 minutes.
  • stringency conditions also depend on the length of the DNA sequences, oligonucleotide probes (such as 10-40 bases) or mixed oligonucleotide probes. If mixed probes are used, it is preferable to use tetramethyl ammonium chloride (TMAC) instead of SSC. See Ausubel, supra.
  • a polypeptide designates any protein or polypeptide encoded by the NRGl gene as disclosed above, respectively.
  • polypeptide designates, within the context of this invention, a polymer of amino acids without regard to the length of the polymer; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide.
  • a fragment of a polypeptide designates any portion of at least 8 consecutive amino acids of a sequence of said protein, preferably of at least about 15, more preferably of at least about 20, further preferably of at least 50, 100, 250, 300 or 350 amino acids.
  • polypeptides which include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide.
  • polypeptides variants which contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
  • the present invention encompasses all polypeptide sequences defined in Figures 2 to 11.
  • the NRGl polypeptide according to this invention has the sequence defined in SEQ ID NO. 1.
  • the NRGl polypeptide according to this invention has the sequence as defined in SEQ ID NO. 2.
  • the NRGl polypeptide according to this invention has the sequence as defined in SEQ ID NO. 3.
  • the NRGl polypeptide according to this invention has the sequence as defined in SEQ ID NO. 7.
  • the NRGl polypeptide according to this invention has the sequence as defined in SEQ ID NO.10.
  • epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags.
  • Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BPl 6 protein fusions.
  • a fusion protein also can be engineered to contain a cleavage site located between the NRGl polypeptide-encoding sequence and the heterologous protein sequence, so that the NRGl polypeptide can be cleaved and purified away from the heterologous moiety.
  • a fusion protein can be synthesized chemically, as is known in the art.
  • a fusion protein is produced by covalently linking two polypeptide segments or by Standard procedures in the art of molecular biology.
  • Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises coding sequences for NRGl in proper reading frame with nucleotides encoding the second polypeptide segment and expressing the DNA construct in a host cell, as is known in the art.
  • treat or “treating” as used herein is meant to ameliorate, alleviate symptoms, eliminate the causation of the symptoms either on a temporary or permanent basis, or to prevent or slow the appearance of symptoms of the named disorder or condition.
  • treatment as used herein also encompasses the term “prevention of the disorder”, which is, e.g., manifested by delaying the onset of the symptoms of the disorder to a medically significant extent. Treatment of the disorder is, e.g., manifested by a decrease in the symptoms associated with the disorder or an amelioration of the reoccurrence of the symptoms of the disorder.
  • modulated or modulation or regulated or “regulation” as used herein refer to both upregulation [i.e., activation or stimulation (e.g., by agonizing or potentiating)] and downregulation [i.e., inhibition or suppression (e.g., by antagonizing, decreasing or inhibiting)].
  • oligonucleotides and “polynucleotides” include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form.
  • nucleotide is used herein as an adjective to describe compounds comprising RNA, DNA, or RNA/DNA hybrid sequences of any length in single-stranded or duplex form.
  • nucleotide is also used herein as a noun to refer to individual nucleotides or varieties of nucleotides, meaning a compound, or individual unit in a larger nucleic acid compound, comprising a purine or pyrimidine, a ribose or deoxyribose sugar moiety, and a phosphate group, or phosphodiester linkage in the case of nucleotides within an oligonucleotide or polynucleotide.
  • nucleotide is also used herein to encompass "modified nucleotides" which comprise at least one modifications (a) an alternative linking group, (b) an analogous form of purine, (c) an analogous form of pyrimidine, or (d) an analogous sugar, for examples of analogous linking groups, purine, pyrimidines, and sugars see for example PCT publication No. WO95/04064, the disclosure of which is incorporated herein by reference.
  • the polynucleotides of the invention are preferably comprised of greater than 50% conventional deoxyribose nucleotides, and most preferably greater than 90% conventional deoxyribose nucleotides.
  • the polynucleotide sequences of the invention may be prepared by any known method, including synthetic, recombinant, ex vivo generation, or a combination thereof, as well as utilizing any purification methods known in the art.
  • isolated requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally- occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that the vector or composition is not part of its natural environment.
  • primer denotes a specific oligonucleotide sequence, which is complementary to a target nucleotide sequence and used to hybridize to the target nucleotide sequence.
  • a primer serves as an initiation point for nucleotide polymerization catalyzed by either DNA polymerase, RNA polymerase or reverse transcriptase.
  • Typical primers of this invention are single-stranded nucleic acid molecules of about 6 to 50 nucleotides in length, more preferably of about 8 to about 40 nucleotides in length, typically of about 16 to 25.
  • the Tm is typically of about 60°C or more.
  • the sequence of the primer can be derived directly from the sequence of the target gene. Perfect complementarity between the primer sequence and the target gene is preferred, to ensure high specificity. However, certain mismatch may be tolerated.
  • probe denotes a defined nucleic acid segment (or nucleotide analog segment, e.g., polynucleotide as defined herein) which can be used to identify a specific polynucleotide sequence present in samples, said nucleic acid segment comprising a nucleotide sequence complementary of the specific polynucleotide sequence to be identified.
  • Probes of this invention typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 750, more preferably of between 15 and 600, typically of between 20 and 400.
  • the sequence of the probes can be derived from the sequences of the NRGl gene sequence.
  • the probe may contain nucleotide substitutions and/or chemical modifications, e.g., to increase the stability of hybrids or to label the probe. Typical examples of labels include, without limitation, radioactivity, fluorescence, luminescence, etc.
  • complementary or “complement thereof are used herein to refer to the sequences of polynucleotides that are capable of forming Watson & Crick base pairing with another specified polynucleotide throughout the entirety of the complementary region. This term is applied to pairs of polynucleotides based solely upon their sequences and not any particular set of conditions under which the two polynucleotides would actually bind.
  • non-human animal refers to any non-human vertebrate, birds and more usually mammals, preferably primates, farm animals such as swine, goats, sheep, donkeys, and horses, rabbits or rodents, more preferably rats or mice.
  • animal is used to refer to any vertebrate, preferable a mammal. Both the terms “animal” and “mammal” expressly embrace human subjects unless preceded with the term "non-human”.
  • twin and phenotype are used interchangeably herein and refer to any clinically distinguishable, detectable or otherwise measurable property of an organism such as symptoms of, or susceptibility to a disease for example.
  • the terms “trait” or “phenotype” are used herein to refer to symptoms of, or susceptibility to inflammatory disorder; or to refer to an individual's response to an agent acting on inflammatory disorder; or to refer to symptoms of, or susceptibility to side effects to an agent acting on inflammatory disorder.
  • allele refers to one of the variant forms of a biallelic or multiallelic alteration, differing from other forms in its nucleotide sequence. Typically the first identified allele is designated as the original allele whereas other alleles are designated as alternative alleles. Diploid organisms may be homozygous or heterozygous for an allelic form.
  • polymorphism refers to the occurrence of two or more alternative genomic sequences or alleles between or among different genomes or individuals. "Polymorphic” refers to the condition in which two or more variants of a specific genomic sequence can be found in a population.
  • a "polymorphic site” is the locus at which the variation occurs.
  • a polymorphism may comprise a substitution, deletion or insertion of one or more nucleotides.
  • a single nucleotide polymorphism is a single base pair change. Typically a single nucleotide polymorphism is the replacement of one nucleotide by another nucleotide at the polymorphic site.
  • SNP single nucleotide polymorphism
  • antibody encompasses monoclonal and polyclonal antibodies, chimeric, humanized, fully human, bispecific or multispecific antibodies as well as fragments thereof such as single chain antibodies (scFv) or domain antibodies, as further explained below.
  • the term "selective" binding indicates that the antibodies preferentially bind the target polypeptide or epitope, i.e., with a higher affinity than any binding to any other antigen or epitope. In other words, binding to the target polypeptide can be discriminated from non-specific binding to other antigens.
  • Antibodies of this invention may be monoclonal or polyclonal antibodies, or fragments or derivative thereof having substantially the same antigen specificity.
  • the term fragment includes any binding portion of an antibody, such as Fab, F(ab')2, CDR domains, etc.
  • Derivatives include human or humanized antibodies, polyfunctional antibodies, single-chain antibodies (e.g., ScFv), etc. Methods for producing antibodies, fragments or derivatives thereof are well known in the art, including immunization of an animal and collection of serum (polyclonal) or spleen cells (to produce hybridomas by fusion with appropriate cell lines).
  • Harlow et al (Antibodies: A laboratory Manual, CSH Press, 1988) or in Kohler et al (Nature 256 (1975) 495), incorporated therein by reference. Briefly, these methods comprise immunizing an animal with the antigen, subsequently recovering spleen cells and fusing these cells with immortalized cells, such as myeloma cells, to produce hybridomas. Hybrodimas producing the desired monoclonal antibodies can be selected by limit dilutions. Antibodies may also be produced by selection of combinatorial libraries of immunoglobulins, as disclosed for instance in Ward et al (Nature 341 (1989) 544).
  • the antibodies may be coupled to heterologous moieties, such as toxins, labels, drugs or other therapeutic agents, covalently or not, either directly or through the use of coupling agents or linkers.
  • heterologous moieties such as toxins, labels, drugs or other therapeutic agents
  • the present invention provides novel means and methodologies for detecting or diagnosing multiple sclerosis, systemic lupus erythematosus, psoriasis and related disorders in a human subject.
  • the present methods may be implemented at various development stages of said pathologies, including early, pre-symptomatic stages, and late stages, in adults, children and pre-birth.
  • the invention is suited to determine the prognosis, to assess a predisposition to or a risk of development of pathology, to characterize the status of a disease or to define the most appropriate treatment regimen for a patient.
  • a particular object of this invention resides in a method of detecting the presence of or predisposition to multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in a subject, the method comprising detecting the presence of an alteration in a NRGl gene or polypeptide in a sample from the subject, the presence of such an alteration being indicative of the presence of or predisposition to multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in said subject.
  • Another object of this invention relates to methods of assessing the response of a subject to a treatment of multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder, the methods comprising detecting the presence of an alteration in a NRGl gene or polypeptide in a sample from the subject, the presence of such an alteration being indicative of a responder subject.
  • interferon and "interferon-beta (IFN-beta)", as used herein, are intended to include fibroblast interferon in particular of human origin, as obtained by isolation from biological fluids or as obtained by DNA recombinant techniques from prokaryotic or eukaryotic host cells, as well as its salts, functional derivatives, variants, analogs and active fragments.
  • IFN-beta suitable in accordance with the present invention is commercially available e.g. as Rebif® (Serono), Avonex® (Biogen) or Betaferon® (Schering).
  • Rebif® Sterotono
  • Avonex® Biogen
  • Betaferon® Schering
  • the use of interferons of human origin is also preferred in accordance with the present invention.
  • the term interferon, as used herein, is intended to encompass salts, functional derivatives, variants, analogs and active fragments thereof.
  • Rebif® (recombinant human interferon-) is the latest development in interferon therapy for multiple sclerosis (MS) and represents a significant advance in treatment.
  • Rebif® is interferon (IFN)-beta Ia, produced from mammalian cell lines. It was established that interferon beta- Ia given subcutaneously three times per week is efficacious in the treatment of Relapsing-Remitting Multiple Sclerosis (RRMS).
  • Interferon beta- Ia can have a positive effect on the long-term course of MS by reducing number and severity of relapses and reducing the burden of the disease and disease activity as measured by MRI.
  • IFN is recombinant IFN- ⁇ Ib produced in E. CoIi, commercially available under the trademark Betaseron
  • IFN may preferably be administered sub-cutaneously every second day at a dosage of about of 250 to 300 g or 8 MIU to 9.6 MIU per person.
  • IFN is recombinant IFN- ⁇ Ia, produced in Chinese Hamster Ovary cells (CHO cells), commercially available under the trademark Avonex
  • IFN may preferably be administered intra-muscularly once a week at a dosage of about of 3Og to 33 g or 6 MIU to 6.6 MIU per person.
  • Quinacrine in a dosage of 100 mg per day, can be added without increasing the risk of retinopathy, or the patient can be switched to chloroquine HCl (Aralen), in a dosage of 250 mg per day.
  • chloroquine HCl Alen
  • lupus nephritis are severe. Patients with milder forms, including mesangial glomerulonephritis and focal proliferative glomerulonephritis, may respond to corticosteroid therapy alone or with steroid-sparing drugs such as azathioprine. Cyclophosphamide (Cytoxan) is more effective than corticosteroids alone for the treatment of severe forms of lupus nephritis (diffuse proliferative glomerulonephritis). One of the major complications of systemic lupus erythematosus is premature or accelerated atherosclerosis.
  • Corticosteroid treatment increases the levels of cardiovascular risk factors, including weight, blood pressure, cholesterol and homocysteine levels.
  • Patients with systemic lupus erythematosus who have already had a manifestation of antiphospholipid antibody syndrome require treatment. Patients who have had venous or arterial thrombosis appear to benefit from maintenance therapy with high- intensity (International Normalized Ratio of 3 to 4) warfarin (Coumadin).
  • the most frequent hematologic manifestation of systemic lupus erythematosus is anemia, usually normochromic normocystic, reflecting chronic illness.
  • Anemia in patients with systemic lupus erythematosus is most often associated with chronic disease or is related to iron deficiency.
  • Classic autoimmune hemolytic anemia can present acutely (and severely) or as a chronic condition. Severe hemolytic anemia is treated initially with intravenous methylprednisolone, 1,000 mg per day for three days.
  • Treatment approaches for psoriasis depend on the severity of the symptoms. In mild forms of psoriasis, topical treatments are applied, such as vitamin D3 analogs, corticosteroids, retinoids and coal tar. Oily bath solutions and moisturizers have a soothing effect on the skin.
  • Moderate to severe psoriasis is usually treated with light therapy. This includes therapy by sunlight or Ultraviolet B. Also available are combination therapies combining oral or topical administration of Psoralen with exposure to Ultraviolet A. Light therapy may also be combined with administration of retinoids.
  • efalizumab (marketed under the tradename Raptiva) is a humanised monoclonal antibody, which is recombinantly produced in mammalian cells. It inhibits activation of T cells and reduces inflammation in psoriasis.
  • alteration in a NRGl gene or polypeptide may be any nucleotide or amino acid alteration associated to multiple sclerosis, systemic lupus erythematosus or a related disease.
  • a susceptibility alteration in the NRGl gene may be any form of mutation(s), deletion(s), rearrangement(s) and/or insertion(s) in the coding and/or non-coding region of the gene, either isolated or in various combination(s). Mutations more specifically include point mutations. Deletions may encompass any region of two or more residues in a coding or non-coding portion of the gene. Typical deletions affect small regions, such as domains (introns) or repeated sequences or fragments of less than about 50 consecutive base pairs, although larger deletions may occur as well. Insertions may encompass the addition of one or several residues in a coding or non-coding portion of the gene. Insertions may typically comprise an addition of between 1 and 50 base pairs in the gene.
  • Rearrangements include for instance sequence inversions.
  • An alteration in the NRGl gene may also be an aberrant modification of the polynucleotide sequence, such as of the methylation pattern of the genomic DNA, allelic loss of the gene or allelic gain of the gene.
  • the alteration may be silent (i.e., create no modification in the amino acid sequence of the protein), or may result, for instance, in amino acid substitutions, frameshift mutations, stop codons, RNA splicing, e.g. the presence of a non-wild type splicing pattern of a messenger RNA transcript, or RNA or protein instability or a non-wild type level of the NRGl polypeptide.
  • the alteration may result in the production of a polypeptide with altered function or stability, or cause a reduction or increase in protein expression levels.
  • Typical susceptibility alterations are single nucleotide polymorphisms (SNPs).
  • NRGl gene which are associated with multiple sclerosis, systemic lupus erythematosus or psoriasis. These mutations are reported in table 2 (tables 2a, 2b, 2c, 2d, 2e). In summary, the association results of the single biallelic alteration frequency analysis show that the NRGl gene is associated with multiple sclerosis, systemic lupus erythematosus and psoriasis.
  • the most preferred alterations linked to SLE or psoriasis are those SNP for which the Mantel-Haenszel statistical test was significant. This means that the allele frequency difference between cases and controls is significant for these SNPs when the two SLE or psoriasis sets of data are analysed simultaneously and stratified by study populations. For MS, the Mantel-Haenszel statistical test was not significant for any of the SNPs; this means, that different SNPs are linked to MS in the different populations.
  • Base 1 base number 119199117 of chromosome 3 to base number 117011800
  • a preferred embodiment of the present invention comprises the detection of the presence of a susceptibility alteration as disclosed in Tables 2a, 2b or 2c in the NRGl gene or RNA sequence of a subject, more particularly the detection of at least one alteration as disclosed in Table 2d or 2e, or any combination thereof.
  • Another preferred object of this invention is a method of detecting the presence of or predisposition to psoriasis or a related disorder in a subject, the method comprising detecting the presence or absence of the associated susceptibility alteration according to table 2c or 2e in a sample from the subject, the presence of the associated susceptibility alteration being indicative of the presence of or predisposition to psoriasis or a related disorder in said subject.
  • NRGl multiple sclerosis, systemic lupus erythematosus or related diseases
  • additional susceptibility alterations can be identified within said gene or polypeptide, e.g., following the methodology disclosed in the examples.
  • the presence of an alteration in the NRGl gene may be detected by any technique known per se to the skilled artisan (reviewed by Kwok et al., 2003), including sequencing, pyrosequencing, selective hybridisation, selective amplification and/or mass spectrometry including matrix-assisted laser desorption/ionization time-of- flight mass spectrometry (MALDI-TOF MS) (Gut et al., 2004).
  • the alteration is detected by selective nucleic acid amplification using one or several specific primers.
  • the alteration is detected by selective hybridization using one or several specific probes.
  • Further techniques include gel electrophoresis-based genotyping methods such as PCR coupled with restriction fragment length polymorphism analysis, multiplex PCR, oligonucleotide ligation assay, and minisequencing; fluorescent dye-based genotyping technologies such as oligonucleotide ligation assay, pyrosequencing, single-base extension with fluorescence detection, homogeneous solution hybridization such as TaqMan, and molecular beacon genotyping; rolling circle amplification and Invader assays as well as DNA chip-based microarray and mass spectrometry genotyping technologies (Shi et al., 2001).
  • gel electrophoresis-based genotyping methods such as PCR coupled with restriction fragment length polymorphism analysis, multiplex PCR, oligonucleotide ligation assay, and minisequencing
  • fluorescent dye-based genotyping technologies such as oligonucleotide ligation assay, pyrosequencing, single-base extension with fluorescence detection, homo
  • RNA expression of altered genes can be quantified by methods known in the art such as subtractive hybridisation, quantitative PCR, TaqMan, differential display reverse transcription PCR, serial, partial sequencing of cDNAs (sequencing of expressed sequenced tags (ESTs) and serial analysis of gene expression (SAGE)), or parallel hybridization of labeled cDNAs to specific probes immobilized on a grid (macro- and microarrays and DNA chips.
  • Particular methods include allele-specific oligonucleotide (ASO), allele-specific amplification, fluorescent in situ hybridization (FISH) Southern and Northern blot, and clamped denaturing gel electrophoresis.
  • Protein expression analysis methods include 2-dimensional gel- electrophoresis, mass spectrometry and antibody microarrays (Freeman et al., 2004 and Zhu et al., 2003).
  • Amplification may be performed according to various techniques known in the art, such as by polymerase chain reaction (PCR), ligase chain reaction (LCR) and strand displacement amplification (SDA). These techniques can be performed using commercially available reagents and protocols.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • a preferred technique is allele-specific PCR.
  • Nucleic acid primers useful for amplifying sequences from the NRGl gene are able to specifically hybridize with a portion of the NRGl gene that either flanks or overlaps with a susceptibility alteration.
  • the primer sequence overlaps with the alteration when said alteration is contained within the sequence of the NRGl gene to which the primer hybridizes.
  • the primer sequence flanks the alteration when the primer hybridizes with a portion of the NRGl gene that is preferably located at a distance below 300 bp of said alteration, even more preferably below 250, 200, 150, 100, 50, 40, 30 or 20 bp from said alteration.
  • the primer hybridizes with a portion of the NRGl gene that is at 5, 4, 3, 2, 1 bp distance or immediately adjacent to said alteration.
  • the methods involve the use of a nucleic acid probe specific for a NRGl or altered NRGl gene, followed by the detection of the presence of a hybrid.
  • the probe may be used in suspension or immobilized on a substrate or support.
  • the probe is typically labelled to facilitate detection of hybrids.
  • Treatments include, for instance, lysis (e.g., mechanical, physical, chemical, etc.), centrifugation, etc.
  • the nucleic acids and/or polypeptides may be pre- purified or enriched by conventional techniques, and/or reduced in complexity. Nucleic acids and polypeptides may also be treated with enzymes or other chemical or physical treatments to produce fragments thereof. Considering the high sensitivity of the claimed methods, very few amounts of sample are sufficient to perform the assay.
  • the finding of an altered NRGl gene or polypeptide in the sample is indicative of the presence, predisposition or stage of progression of multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in the subject.
  • a related disorder in the subject.
  • one only of the above-disclosed susceptibility alterations is assessed, or several of them, in combination(s).
  • kits for the identification of a genetic polymorphism pattern at the NRGl gene associated with increased risk of the presence of or predisposition to multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in a subject comprising:
  • (b) means for determining a genetic polymorphism pattern for the NRGl gene.
  • the present invention also provides novel targets and methods for the screening of drug candidates or leads.
  • These screening methods include binding assays and/or functional assays, and may be performed in vitro, in cell systems or in animals.
  • Another object of this invention resides in methods of selecting biologically active compounds, said methods comprising contacting a candidate compound with a NRGl gene or polypeptide, and selecting compounds that bind said gene or polypeptide.
  • a “biologically active” compound denotes any compound having biological activity in a subject, preferably therapeutic activity, and further preferably a compound that can be used for treating multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder, or as a lead to develop drugs for treating multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder.
  • a “biologically active” compound preferably is a compound that modulates the activity of NRGl.
  • this invention relates to a method of screening, selecting or identifying active compounds, particularly compounds active on multiple sclerosis, systemic lupus erythematosus, psoriasis or related disorders, the method comprising contacting a test compound with a recombinant host cell comprising a reporter construct, said reporter construct comprising a reporter gene under the control of a NRGl gene promoter, and selecting the test compounds that modulate (e.g. stimulate or reduce, preferably stimulate) expression of the reporter gene.
  • active compounds particularly compounds active on multiple sclerosis, systemic lupus erythematosus, psoriasis or related disorders
  • the modulator is a NRGl fusion protein.
  • a fusion protein of NRGl may e.g. comprise an immunoglobulin fusion, i.e. a fused protein comprising all or part of a NRGl protein, which is fused to all or a portion of an immunoglobulin.
  • Methods for making immunoglobulin fusion proteins are well known in the art, such as the ones described in WO 01/03737, for example. The person skilled in the art will appreciate that the resulting fusion protein of the invention substantially retains the biological activity of NRGl, which can be measured in in vitro assays.
  • the modulator is an inhibitor or antagonist of a NRGl polypeptide.
  • a further object of this invention resides in a pharmaceutical composition
  • a pharmaceutical composition comprising a functional NRGl polypeptide or a nucleic acid encoding a NRGl polypeptide or a vector encoding the same, and a pharmaceutically acceptable diluent or carrier.
  • Selected for the SLE study were individuals who fulfilled at least 4 of the 11 ACR criteria.
  • Autoprimer.com automatically optimizes the grouping of the alterations by extension mix and appends tag sequences to the 5' ends of the SNP-IT primers, which are complementary to the tags immobilized on the microarray plate.
  • Affymetrix genotyping technology Use of Affymetrix genotyping technology have been described in Kennedy, G.C., et al. Nature Biotechnology 21, 1233-1237, 2003 (Large-scale genotyping of complex DNA). ; Liu, W.M., et al. Bioinformatics 19, 2397-2403, 2003. (Algorithms for Large Scale Genotyping Microarrays) ;Matsuzaki, H., et al. Genome Research 3, 414-25, 2004. (Parallel Genotyping of over 10,000 SNPs using a One Primer Assay on a High Density Oligonucleotide Array) ;Paez, J.G., et al. Nucleic Acids Research 32(9), 2004.
  • Hybridization signals are generated in a three step signal amplification process: lO ⁇ g/mL streptavidin R-phycoerythrin (SAPE) conjugate (Molecular Probes) is added to the biotinylated targets hybridized to the oligonucleotide probes, and washed with 6X SSPE and 0.01% Tween-20 at 25 °C; followed by the addition of 5 ⁇ g/mL biotinylated goat anti-streptavidin (Vector) to increase the effective number of biotin molecules on the target; and finally SAPE is added once again and washed extensively with 6X SSPE and 0.01% Tween-20 at 30 °C.
  • SAPE streptavidin R-phycoerythrin conjugate
  • the SAPE and antibody were added to arrays in 6X SSPE, IX Denhardt's solution and 0.01% Tween-20 at 25 °C for 10 minutes each. Following the final wash, the arrays are kept in Holding buffer (10OmM MES, IM [Na+], 0.01% Tween-20). The washing and staining procedures are run on Affymetrix fluidics stations. Arrays are scanned using GCS3000 scanners with
  • Genomic Control (Devlin andRoeder 1999): given that in the presence of population substructure, the standard chi-square statistic is inflated by a multiplicative factor, which is proportional to the degree of stratification, we can estimate and incorporate this multiplicative factor (lambda) into the disease - susceptibility alteration association tests (by rescaling the chi-square statistic) to correct for background population differences.

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Abstract

The present invention relates, generally, to methods and compositions for detecting or treating inflammatory disorders, such as multiple sclerosis, systemic lupus erythematosus or psoriasis. The present invention more particularly discloses the identification of human genes, which can be used for the diagnosis, prevention, and treatment of multiple sclerosis, systemic lupus erythematosus, psoriasis and related disorders, as well as for the screening of therapeutically active drugs. The invention further discloses specific polymorphisms, splice variants or alleles of the NRG1 gene that are related to multiple sclerosis, systemic lupus erythematosus and psoriasis, as well as diagnostic tools and kits based on these polymorphisms. The invention can be used in the diagnosis of or predisposition to, detection, prevention and/or treatment of multiple sclerosis, systemic lupus erythematosus, psoriasis and related disorders.

Description

COMPOSITIONS AND METHODS FOR TREATING INFLAMMATORY DISORDERS
FIELD OF THE INVENTION
The present invention relates, generally, to methods and compositions for detecting or treating inflammatory disorders. The inflammatory disorder may be an inflammatory CNS disorder such as multiple sclerosis (MS), an inflammatory disorder involving multiple organ systems as in the case of systemic lupus erythematosus (SLE) or an inflammatory skin disorder such as psoriasis. The present invention more particularly relates to the human NRGl gene, which can be used for the diagnosis, prevention and treatment of multiple sclerosis, systemic lupus erythematosus, psoriasis and related disorders, as well as for the screening of therapeutically active drugs. The invention further discloses specific polymorphisms, splice variants or alleles of the NRGl gene that are related to inflammatory disorders, such as multiple sclerosis, systemic lupus erythematosus or psoriasis, as well as diagnostic tools and kits based on these susceptibility alterations. Thus, the invention can be used in the diagnosis or detection of the presence, risk or predisposition to, as well as in the prevention and/or treatment of multiple sclerosis, systemic lupus erythematosus, psoriasis and related disorders.
BACKGROUND OF THE INVENTION
Multiple sclerosis (MS) is the most known chronic inflammatory demyelinating disease of the central nervous system in humans. The onset of the disease typically occurs during ages 20 to 40. Women are affected approximately twice as often as men.
Over time, MS may result in the accumulation of various neurological disabilities. Clinical disability in MS is presumed to be a result of repeated inflammatory injury with subsequent loss of myelin and axons, leading to tissue atrophy.
MS is manifested in physical symptoms (relapses and disability progression), Central Nervous System (CNS) inflammation, brain atrophy and cognitive impairment. Presenting symptoms include focal sensory deficits, focal weakness, visual problems, imbalance and fatigue. Sexual impairment and sphincter dysfunction may occur. Approximately half of the patients with MS may experience cognitive impairment or depression.
MS is now considered to be a multi-phasic disease and periods of clinical quiescence (remissions) occur between exacerbations. Remissions vary in length and may last several years but are infrequently permanent.
Four courses of the disease are individualized: relapsing-remitting (RR), secondary progressive (SP), primary progressive (PP) and progressive relapsing (PR) multiple sclerosis. More than 80% of patients with MS will initially display a RR course with clinical exacerbation of neurological symptoms, followed by a recovery that may or may not be complete (Lublin and Reingold, Neurology, 1996, 46:907-911).
During RRMS, accumulation of disability results from incomplete recovery from relapses. Approximately, half of the patients with RRMS switch to a progressive course, called SPMS, 10 years after the disease onset. During the SP phase, worsening of disability results from the accumulation of residual symptoms after exarcerbation but also from insidious progression between exacerbations (Lublin and Reingold above). 10% of MS patients have PPMS which is characterized by insidious progression of the symptoms from the disease onset. Less than 5 % of patients have PRMS and are often considered to have the same prognosis as PPMS. It is suggested that distinct pathogenic mechanisms may be involved in different patient sub-groups and have wide-ranging implications for disease classification (Lassmann et ah, 2001, Trends MoI. Med., 7, 115-121; Lucchinetti et al., Curr. Opin. Neurol, 2001, 14, 259-269).
MS onset is defined by the occurrence of the first neurological symptoms of CNS dysfunction. Advances in cerebrospinal fluid (CSF) analysis and magnetic resonance imaging (MRI) have simplified the diagnostic process and facilitated early diagnostic (Noseworthy et al, The New England Journal of Medicine, 2000, 343, 13, 938-952). The International Panel on the Diagnosis of MS issued revised criteria facilitating the diagnosis of MS and including MRI together with clinical and para-clinical diagnostic methods (Mc Donald et al, 2001, Ann. Neurol, 50:121-127). Systemic Lupus Erythematosus is a disorder in which auto-antibodies lead to inflammation and damage to various body tissues. Systemic Lupus Erythematosus can affect many parts of the body, including the joints, skin, kidneys, heart, lungs, blood vessels and brain. Although people with the disease may have many different symptoms, some of the most common ones include extreme fatigue, painful or swollen joints (arthritis), unexplained fever, skin rashes and kidney problems. The symptoms of SLE may be mild or serious. Although SLE usually first affects people between the ages of 15 and 45 years, it can occur in childhood or later in life as well. Many more women than men have SLE. SLE is three times more common in African American women than in Caucasian women and is also more common in women of Hispanic, Asian and Native American descent. Onset of the disease is very hard to pinpoint.
SLE is characterized by periods of illness, called flares, and periods of wellness, or remission. At present, there is no cure for SLE, however, symptoms may be treated with drugs {Handout on Health: Systemic Lupus Erythematosus, NIAMS, National Institute of Health, Publication date September 1997, revised August 2003).
Psoriasis is a chronic skin disorder for which there is no known medically proven cure. The name of the disease originates from the Greek Psora, which means to to itch and is classed as a non-contagious skin disease.
Clinical hallmarks comprise erythematous plaques covered by silvery scaling and a chronic recurrent course. Histologically, psoriasis is characterized by the hyperproliferation of the epidermis, elongated and prominent blood vessels and a thick perivascular lymphocytic infiltrate. Psoriasis is now considered an auto-immune disease
Although the disease occurs in all age groups, it primarily affects adults. It appears about equally in males and females. Psoriasis occurs when skin cells quickly rise from their origin below the surface of the skin and pile up on the surface before they have a chance to mature. Usually this movement (also called turnover) takes about a month, but in psoriasis it may occur in only a few days. In its typical form, psoriasis results in patches of thick, red (inflamed) skin covered with silvery scales. These patches, which are sometimes referred to as plaques, usually itch or feel sore. They most often occur on the elbows, knees, other parts of the legs, scalp, lower back, face, palms, and soles of the feet, but they can occur on skin anywhere on the body .Psoriasis is considered mild if it affects less than 5% of the surface of the body, moderate if 5-30% of the skin is involved, and severe if the disease affects more than 30% of the body surface.
Psoriasis usually involves the scalp and the extensor surfaces of the limbs especially the elbows and knees.
There are a number of psoriatic subtypes which describe the area of involvement, for example flexural psoriasis (where the skin lesions occur on flexor surfaces such as the groin), or the pattern of cutaneous change, for example psoriasis annularis (psoriasis with lesions occurring in ring shaped patches), or the type of cutaneous lesion, for example pustular psoriasis (where pustules predominate rather than papules, plaques or macules). Psoriatic arthritis, an erosive and usually asymmetrical oligoarthritis, may occur with this chronic recurrent papulosquamous skin disorder.
The symptoms of psoriasis may be mild, moderate or severe. There is currently no cure for psoriasis, but symptoms may be treated, depending on their severity.
Molecules currently used for the treatment of multiple sclerosis, systemic lupus erythematosus and psoriasis in their various forms may have side effects and may act only against the symptoms of the disease. Consequently, there is a strong need for new molecules without or with less associated side effects that are directed against novel targets. Therefore, there is a need to identify proteins involved in the diseases, thereby providing new targets allowing new screenings for drugs, resulting in new drugs that are efficient in treatment of these serious inflammatory diseases and related disorders.
Furthermore, there is also a need for new diagnostic tools. Affected patients may not realize that they are ill, or they may be afraid to seek help; family members sometimes hope the problem will simply disappear or cannot persuade the patient to seek treatment; clinicians may hesitate to prescribe medications when the diagnosis is uncertain because of potential side effects. Indeed, at the first manifestation of the disease, multiple sclerosis, systemic lupus erythematosus and psoriasis may be difficult to diagnose with certainty. Accordingly, there is a need for new methods for detecting a susceptibility to multiple sclerosis, systemic lupus erythematosus, psoriasis and related disorders. SUMMARY OF THE INVENTION
The present invention now discloses novel approaches to the diagnosis and treatment of multiple sclerosis, systemic lupus erythematosus, psoriasis and related disorders, as well as for the screening of therapeutically active drugs. The invention more specifically demonstrates that alterations in the NRGl gene are associated with the development of multiple sclerosis and systemic lupus erythematosus. NRGl, and altered forms of NRGl in particular, represent novel targets for therapeutic intervention against MS, SLE, psoriasis and related pathologies .
A first aspect of this invention thus resides in the use of a NRGl gene or polypeptide as a target for the screening of candidate drug modulators, particularly candidate drugs active against multiple sclerosis, systemic lupus erythematosus, psoraisis and related disorders.
A further aspect of this invention resides in methods of screening of compounds for therapy of multiple sclerosis, systemic lupus erythematosus, psoriasis or related disorders, comprising determining the ability of a compound to bind a NRGl gene or polypeptide, or a fragment thereof, particularly of an allele of said gene or polypeptide that is associated with multiple sclerosis or a related disorder, or a fragment thereof.
A further aspect of this invention resides in methods of screening of compounds for therapy of multiple sclerosis, systemic lupus erythematosus, psoriasis or related disorders, comprising testing for modulation of the activity of a NRGl gene or polypeptide, particularly an allele of said gene or polypeptide that is associated with multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder, or a fragment thereof.
Another aspect of this invention resides in a method of assessing the presence of or predisposition to multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in a subject, comprising determining (in vitro or ex vivo) the presence of an alteration (e.g., a susceptibility mutation or allele) in a NRGl gene or polypeptide in a sample from the subject, the presence of such an alteration being indicative of the presence of or predisposition to multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in said subject.
A further aspect of this invention relates to the use of a modulator of a NRGl gene or polypeptide, preferably an agonist thereof, for the preparation of a medicament for treating or preventing multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in a subject, as well as to corresponding methods of treatment.
A further aspect of this invention relates to methods of treating multiple sclerosis, systemic lupus erythematosus, psoriasis or related disorders in a subject through a modulation of
NRGl gene or polypeptide expression or activity, preferably through an activation or restoration thereof. Such treatments use, for instance, a NRGl polypeptide, a NRGl DNA sequence (including antisense sequences, RNAi), antibodies against NRGl polypeptides, ligands of NRGl or drugs that modulate, preferably mimic or stimulate, NRGl expression or activity. The invention particularly relates to methods of treating individuals having disease-associated alleles of the NRGl gene such as further described herein, in particular in Tables 2a to 2e.
A further aspect of this invention relates to the screening of alteration(s) associated with multiple sclerosis, systemic lupus erythematosus, psoriasis or related disorders in the NRGl gene locus in patients. Such screenings are useful for diagnosing the presence, risk or predisposition to multiple sclerosis, systemic lupus erythematosus, psoriasis and related disorders, and/or for assessing the efficacy of a treatment of such disorders.
A further aspect of this invention includes nucleic acid probes and primers that allow specific detection of susceptibility alterations in a NRGl gene or RNA through selective hybridization or amplification. The invention also encompasses particular nucleic acids, vectors and recombinant cells, as well as kits or solid phase bound nucleic acids or proteins such as DNA or protein arrays or chips suitable for implementing the above detection, screening or treatment methods. In particular, the invention also discloses and encompasses susceptibility alterations in the NRGl nucleic acids and polypeptides that are associated with multiple sclerosis, systemic lupus erythematosus, psoriasis and related disorders. Examples of such susceptibility alterations are more particularly selected from the SNPs as listed in Table 2a, 2b, 2c, 2d and 2e.
The invention can be used in the diagnosis of predisposition to, detection, prevention and/or treatment of multiple sclerosis, systemic lupus erythematosus, psoriasis and related disorders in any mammalian subjects, particularly human patients.
In a further aspect, the invention relates to antibodies, which specifically bind to the NRGl polypeptide.
DESCRIPTION OF THE FIGURES
Figure 1: Genomic organization of human NRGl on chromosome 8 between position 31617043 and 32741561 in plus direction of the chromosome (ENSEMBL version 30.35c). The different exons and introns that can be included in the NRGl isoforms according to Swissprot are described.
Figure 2: Represents the isoform 1 of NRGl according to Swissprot. It has the Refseq accession number NP 039258. This polypeptide sequence is referred to herein as SEQ ID No. 1. This isoform has a transmembrane sequence which is considered as an internal signal sequence and may be cleaved off to give a secreted NRGl protein.
Figure 3: Represents the isoform 2 of NRGl according to Swissprot, which also has the Swissprot definition AlphalA. It has no Refseq accession number. This polypeptide sequence is referred to herein as SEQ ID No. 2. This isoform has a transmembrane sequence which is considered as an internal signal sequence and may be cleaved off to give a secreted NRGl protein.
Figure 4: Represents the isoform 3 of NRGl according to Swissprot, which also has the Swissprot definition Alpha2B. It has the Refseq accession number NP 039254. This polypeptide sequence is referred to herein as SEQ ID No. 3. This isoform has a transmembrane sequence which is considered as an internal signal sequence and may be cleaved off to give a secreted NRGl protein.
Figure 5: Represents the isoform 4 of NRGl according to Swissprot, which also has the Swissprot definition Alpha3. It has no Refseq accession number. This polypeptide sequence is referred to herein as SEQ ID No. 4.
Figure 6: Represents the isoform 6 of NRGl according to Swissprot, which also has the Swissprot definition Betal. Apart from one mutation it corresponds to Refseq accession number NP 039250. This polypeptide sequence is referred to herein as SEQ ID No. 5. This isoform has a transmembrane sequence which is considered as an internal signal sequence and may be cleaved off to give a secreted NRGl protein.
Figure 7: Represents the isoform 7 of NRGl according to Swissprot, which also has the Swissprot definition Beta2. Apart from two mutations it corresponds to Refseq accession number NP 039251. This polypeptide sequence is referred to herein as SEQ ID No. 6. This isoform has a transmembrane sequence which is considered as an internal signal sequence and may be cleaved off to give a secreted NRGl protein.
Figure 8: Represents the isoform 8 of NRGl according to Swissprot, which also has the Swissprot definitions Beta3 and GGFHFBl. It has the Refseq accession number NP 039255. This polypeptide sequence is referred to herein as SEQ ID No. 7.
Figure 9: Represents the isoform 9 of NRGl according to Swissprot, which also has the Swissprot definitions GGF2 and GGFHPP2. Apart from one mutation it corresponds to Refseq accession number NP 039256. This polypeptide sequence is referred to herein as SEQ ID No. 8. This isoform is a secreted protein.
Figure 10: Represents the isoform 10 of NRGl according to Swissprot, which also has the Swissprot definition SMDF (sensory and motor neuron-derived factor isoform). It has the Refseq accession number NP 039253. This polypeptide sequence is referred to herein as SEQ ID No. 9. This isoform is a secreted protein. Figure 11: Represents an isoform of NRGl having the Refseq accession number NP 004486. It has no Swissprot number. This polypeptide sequence is referred to herein as SEQ ID No. 10.
DETAILED DESCRIPTION OF THE INVENTION
The present invention stems from association studies conducted on different MS, SLE and psoriasis populations, using a number of random alterations. The results of these studies show that the NRGl gene is strongly associated with multiple sclerosis, systemic lupus erythematosus and psoriasis and that new and validated (biallelic) susceptibility alterations located in said gene or corresponding RNAs are associated with multiple sclerosis,
systemic lupus erythematosus, psoriasis and related disorders.
The present invention thus provides novel means and methods to identify compounds useful in the treatment of multiple sclerosis, systemic lupus erythematosus, psoriasis and related disorders. The invention further provides novel approaches to the detection, diagnosis and monitoring of multiple sclerosis, systemic lupus erythematosus, psoriasis or related disorders in a subject, as well as for genotyping of inflammatory patients, in particular patients having MS, SLE or psoriasis. The invention further provides novel approaches to the prevention and treatment of multiple sclerosis, systemic lupus erythematosus, psoriasis or related disorders in a subject.
DEFINITIONS The term "multiple sclerosis" may be defined as in the DSM-IV classification (Diagnosis and Statistical Manual of Inflammatory CNS Disorders, Fourth Edition, American Psychiatric Association, Washington D.C., 1994). The term "systemic lupus erythematosus" may be defined as by Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF et al. "The 1982 revised criteria for the classification of Systemic lupus erythematosus" Arthritis Rheum 1982;25: 1271 -1277.
The term "psoriasis" may be defined as by M. A. de Rie, A. Y. Goedkoop, & J.D. Bos: Overview of psoriasis, Dermatol Ther. 2004;17(5):341-9 or T.J. Lawley, K.B. Yancey, approach to the patient with skin disorder, in Harrison's Principles of internal medicine, 16th Edition, 283 - 297. The term "inflammatory disorder" includes in particular demyelinating inflammatory CNS disorders, such as for example MS and related disorders, inflammatory skin disorders, such as for example psoriasis, and inflammatory disorders involving multiple organ systems, such as for example SLE and related disorders.
Disorders related to MS include progressive multifocal leukoencephalopathy (PML), acute disseminated encephalomyelitis (ADEM), acute demyelinating polyneuropathy (Guillain Barre syndrome), chronic inflammatory demyelinating neuropathy, Marchivafa-Bignami disease, central pontine myelinolysis, Devic syndrome, BaIo disease, HIV- or HTLV- myelopathy or a secondary demyelinating disorder such as CNS lupus erythematodes, polyarteritis nodosa, Sjogren syndrome, sarcoidosis or isolated cerebral vasulitis. Disorders related to SLE include cutaneous lupus, Drug-induced lupus, neonatal lupus and disorders such as rheumatoid arthritis, Sjogren's syndrome and scleroderma. Disorders related to Psoriasis include psoriatic arthritis.
It has now surprisingly been found that NRGl is linked to inflammatory disorders such as MS, SLE or psoriasis.
As used in the present application, the term "NRGl" designates the human NRGl gene, as well as variants, analogs and fragments thereof. NRGl, which is also referred to as Heregulin alpha, HRGA, Neu differentiation factor, NDF, glial growth factor 2, GGF2, is located on gene map locus 8p22-pl 1 and belongs to the neuregulin family.
The nucleic and amino acid sequences of NRGl gene or polypeptide are available in the literature. 10 isoforms (isoform 1, isoform 2, isoform 3, isoform 4, isoform 6, isoform 7, isoform 8, isoform 9, isoform 10 and Refseq accession number NP 004486) are known according to Swissprot and Refseq and are described in the Figures 2 to 11.
NRGl has been implicated in human disease. It has been suggested that the isoform 10 may play a role in motor and sensory neuron development; therefore it is also referred to in the abbreviated form SMDF. NRGl is a direct ligand for ERBB3 and ERBB4 tyrosine kinase receptors. NRGl concomitantly recruits ERBBl and ERBB2 coreceptors, resulting in ligand-stimulated tyrosine phosphorylation and activation of the ERBB receptors. The multiple isoforms perform diverse functions such as inducing growth and differentiation of epithelial, glial, neuronal, and skeletal muscle cells; inducing expression of acetylcholine receptor in synaptic vesicles during the formation of the neuromuscular junction; stimulating lobuloalveolar budding and milk production in the mammary gland and inducing differentiation of mammary tumor cells; stimulating Schwann cell proliferation; implication in the development of the myocardium such as trabeculation of the developing heart.
By a genomewide scan of schizophrenia families in Iceland, Stefansson et al. (2002) showed that a schizophrenia locus maps to chromosome 8p, as had been suggested by previous work done in 5 populations. Extensive fine mapping of the 8p locus and haplotype-association analysis, supplemented by a transmission/disequilibrium test, identified NRGl as a candidate gene for schizophrenia. NRGl hypomorphs have fewer functional NMDA (N-Methyl-D-Aspartat) receptors than wildtype mice. Steiansson et al. (Am. J. Hum. Genet. January 2003;72(l):83-87) also demonstrated that the behavioral phenotypes of the NRGl hypomorphs were partially reversible with clozapine, an atypical antipsychotic drug used to treat schizophrenia.
Duchenne muscular dystrophy (DMD) is a fatal disorder caused by absence of dystrophin. Krag et al. (PNAS, September 2004; 101(38):13856-60) tested the ability of NRGl isoform 6 to improve the dystrophic phenotype in the mdx mouse model of DMD. Intraperitoneal injections of the small peptide encoding the epidermal growth factor-like region of heregulin ectodomain for 3 months in vivo resulted in upregulation of utrophin, a marked improvement in the mechanical properties of muscle as evidenced by resistance to eccentric contraction-mediated damage, and a reduction of muscle pathology.
The term "gene" as used herein shall be construed to include any type of coding nucleic acid region, including genomic DNA (gDNA), complementary DNA (cDNA), synthetic or semi- synthetic DNA, any form of corresponding RNA (e.g., mRNA), etc., as well as non coding sequences, such as introns, 5'- or 3 '-untranslated sequences or regulatory sequences (e.g., promoter or enhancer), etc. The term gene particularly includes recombinant nucleic acids, i.e., any non naturally occurring nucleic acid molecule created artificially, e.g., by assembling, cutting, ligating or amplifying sequences. A gene is typically double-stranded, although other forms may be contemplated, such as single-stranded. Genes may be obtained from various sources and according to various techniques known in the art, such as by screening DNA libraries or by amplification from various natural sources. Recombinant nucleic acids may be prepared by conventional techniques, including chemical synthesis, genetic engineering, enzymatic techniques, or a combination thereof. The term "gene" may comprise any and all splicing variants of said gene.
The present invention also encompasses fragments of the NRGl gene. A fragment of a gene designates any portion of at least about 8 consecutive nucleotides of a sequence of said gene, preferably at least about 15, more preferably at least about 25 nucleotides, further preferably of at least 35, 50, 75, 100, 150, 200 or 300 nucleotides. Fragments include more particularly all possible nucleotide length between 8 and 500 nucleotides, preferably between 15 and 300, more preferably between 25 and 200.
The present invention also encompasses DNA sequences, which have at least 40% identity with the NRGl sequence. More preferably, they have at least 50%, at least 60%, at least 70%, at least 80% or, most preferably, at least 90% identity thereto.
Identity reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing the sequences. In general, identity refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of the two polynucleotides or two polypeptide sequences, respectively, over the length of the sequences being compared.
The present invention also encompasses DNA sequences, which hybridize to the complement of the NRGl nucleic acid sequence under moderately stringent conditions or under highly stringent conditions.
The term "stringent conditions" refers to hybridization and subsequent washing conditions, which those of ordinary skill in the art conventionally refer to as "stringent". See Ausubel et ah, Current Protocols in Molecular Biology, supra, Interscience, N.Y., §§6.3 and 6.4 (1987, 1992), and Sambrook et a (Sambrook, J. C, Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
Without limitation, examples of stringent conditions include washing conditions 12-20°C below the calculated Tm of the hybrid under study in, e.g., 2 x SSC and 0.5% SDS for 5 minutes, 2 x SSC and 0.1% SDS for 15 minutes; 0.1 x SSC and 0.5% SDS at 37°C for 30-60 minutes and then, a 0.1 x SSC and 0.5% SDS at 68°C for 30-60 minutes. Those of ordinary skill in this art understand that stringency conditions also depend on the length of the DNA sequences, oligonucleotide probes (such as 10-40 bases) or mixed oligonucleotide probes. If mixed probes are used, it is preferable to use tetramethyl ammonium chloride (TMAC) instead of SSC. See Ausubel, supra.
A polypeptide designates any protein or polypeptide encoded by the NRGl gene as disclosed above, respectively. In this respect, the term "polypeptide" designates, within the context of this invention, a polymer of amino acids without regard to the length of the polymer; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. A fragment of a polypeptide designates any portion of at least 8 consecutive amino acids of a sequence of said protein, preferably of at least about 15, more preferably of at least about 20, further preferably of at least 50, 100, 250, 300 or 350 amino acids. This term also includes post-translational or post-expression modifications of polypeptides, for example, polypeptides which include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide. Also included within the definition are polypeptides variants which contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
The present invention encompasses all polypeptide sequences defined in Figures 2 to 11.
Preferably, the NRGl polypeptide according to this invention has the sequence defined in SEQ ID NO. 1.
In another preferred embodiment, the NRGl polypeptide according to this invention has the sequence as defined in SEQ ID NO. 2.
In another preferred embodiment, the NRGl polypeptide according to this invention has the sequence as defined in SEQ ID NO. 3.
In another preferred embodiment, the NRGl polypeptide according to this invention has the sequence as defined in SEQ ID NO. 4.
In another preferred embodiment, the NRGl polypeptide according to this invention has the sequence as defined in SEQ ID NO. 5.
In another preferred embodiment, the NRGl polypeptide according to this invention has the sequence as defined in SEQ ID NO. 6.
In another preferred embodiment, the NRGl polypeptide according to this invention has the sequence as defined in SEQ ID NO. 7.
In another preferred embodiment, the NRGl polypeptide according to this invention has the sequence as defined in SEQ ID NO. 8. In another preferred embodiment, the NRGl polypeptide according to this invention has the sequence as defined in SEQ ID NO. 9. This polypeptide is secreted and particularly preferred in this invention.
In another preferred embodiment, the NRGl polypeptide according to this invention has the sequence as defined in SEQ ID NO.10.
The present invention also encompasses peptide sequences, which have at least 40% identity with the sequence of SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 3 or SEQ ID NO. 4 or SEQ ID NO. 5 or SEQ ID NO. 6 or SEQ ID NO. 7 or SEQ ID NO. 8 or SEQ ID NO. 9 or SEQ ID NO.10. More preferably, they have at least 50%, at least 60%, at least 70%, at least 80% or, most preferably, at least 90% identity thereto.
Fusion proteins are useful for generating antibodies against a NRGl polypeptide and for use in various assay systems. For example, fusion proteins can be used to identify proteins, which interact with portions of a NRGl polypeptide. Protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.
A NRGl polypeptide fusion protein comprises two polypeptide segments fused together by means of a peptide bond. The first polypeptide segment comprises at least 25, 50, 75, 100, 150, 200, 300, 350 or 372 contiguous amino acids of NRGl. The second polypeptide segment can be a full-length protein or a protein fragment. Proteins commonly used in fusion protein construction include beta-galactosidase, beta-glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT). Additionally, epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BPl 6 protein fusions. A fusion protein also can be engineered to contain a cleavage site located between the NRGl polypeptide-encoding sequence and the heterologous protein sequence, so that the NRGl polypeptide can be cleaved and purified away from the heterologous moiety.
A fusion protein can be synthesized chemically, as is known in the art. Preferably, a fusion protein is produced by covalently linking two polypeptide segments or by Standard procedures in the art of molecular biology. Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises coding sequences for NRGl in proper reading frame with nucleotides encoding the second polypeptide segment and expressing the DNA construct in a host cell, as is known in the art.
The term "treat" or "treating" as used herein is meant to ameliorate, alleviate symptoms, eliminate the causation of the symptoms either on a temporary or permanent basis, or to prevent or slow the appearance of symptoms of the named disorder or condition. The term "treatment" as used herein also encompasses the term "prevention of the disorder", which is, e.g., manifested by delaying the onset of the symptoms of the disorder to a medically significant extent. Treatment of the disorder is, e.g., manifested by a decrease in the symptoms associated with the disorder or an amelioration of the reoccurrence of the symptoms of the disorder.
The terms "modulated" or "modulation" or "regulated" or "regulation" as used herein refer to both upregulation [i.e., activation or stimulation (e.g., by agonizing or potentiating)] and downregulation [i.e., inhibition or suppression (e.g., by antagonizing, decreasing or inhibiting)].
As used interchangeably herein, the term "oligonucleotides", and "polynucleotides" include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form. The term "nucleotide" is used herein as an adjective to describe compounds comprising RNA, DNA, or RNA/DNA hybrid sequences of any length in single-stranded or duplex form. The term "nucleotide" is also used herein as a noun to refer to individual nucleotides or varieties of nucleotides, meaning a compound, or individual unit in a larger nucleic acid compound, comprising a purine or pyrimidine, a ribose or deoxyribose sugar moiety, and a phosphate group, or phosphodiester linkage in the case of nucleotides within an oligonucleotide or polynucleotide. Although the term "nucleotide" is also used herein to encompass "modified nucleotides" which comprise at least one modifications (a) an alternative linking group, (b) an analogous form of purine, (c) an analogous form of pyrimidine, or (d) an analogous sugar, for examples of analogous linking groups, purine, pyrimidines, and sugars see for example PCT publication No. WO95/04064, the disclosure of which is incorporated herein by reference. However, the polynucleotides of the invention are preferably comprised of greater than 50% conventional deoxyribose nucleotides, and most preferably greater than 90% conventional deoxyribose nucleotides. The polynucleotide sequences of the invention may be prepared by any known method, including synthetic, recombinant, ex vivo generation, or a combination thereof, as well as utilizing any purification methods known in the art.
The term "isolated" requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally- occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that the vector or composition is not part of its natural environment.
The term "primer" denotes a specific oligonucleotide sequence, which is complementary to a target nucleotide sequence and used to hybridize to the target nucleotide sequence. A primer serves as an initiation point for nucleotide polymerization catalyzed by either DNA polymerase, RNA polymerase or reverse transcriptase. Typical primers of this invention are single-stranded nucleic acid molecules of about 6 to 50 nucleotides in length, more preferably of about 8 to about 40 nucleotides in length, typically of about 16 to 25. The Tm is typically of about 60°C or more. The sequence of the primer can be derived directly from the sequence of the target gene. Perfect complementarity between the primer sequence and the target gene is preferred, to ensure high specificity. However, certain mismatch may be tolerated.
The term "probe" denotes a defined nucleic acid segment (or nucleotide analog segment, e.g., polynucleotide as defined herein) which can be used to identify a specific polynucleotide sequence present in samples, said nucleic acid segment comprising a nucleotide sequence complementary of the specific polynucleotide sequence to be identified. Probes of this invention typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 750, more preferably of between 15 and 600, typically of between 20 and 400. The sequence of the probes can be derived from the sequences of the NRGl gene sequence. The probe may contain nucleotide substitutions and/or chemical modifications, e.g., to increase the stability of hybrids or to label the probe. Typical examples of labels include, without limitation, radioactivity, fluorescence, luminescence, etc.
The terms "complementary" or "complement thereof are used herein to refer to the sequences of polynucleotides that are capable of forming Watson & Crick base pairing with another specified polynucleotide throughout the entirety of the complementary region. This term is applied to pairs of polynucleotides based solely upon their sequences and not any particular set of conditions under which the two polynucleotides would actually bind.
As used herein, the term "non-human animal" refers to any non-human vertebrate, birds and more usually mammals, preferably primates, farm animals such as swine, goats, sheep, donkeys, and horses, rabbits or rodents, more preferably rats or mice. As used herein, the term "animal" is used to refer to any vertebrate, preferable a mammal. Both the terms "animal" and "mammal" expressly embrace human subjects unless preceded with the term "non-human".
The terms "trait" and "phenotype" are used interchangeably herein and refer to any clinically distinguishable, detectable or otherwise measurable property of an organism such as symptoms of, or susceptibility to a disease for example. Typically the terms "trait" or "phenotype" are used herein to refer to symptoms of, or susceptibility to inflammatory disorder; or to refer to an individual's response to an agent acting on inflammatory disorder; or to refer to symptoms of, or susceptibility to side effects to an agent acting on inflammatory disorder. As used herein, the term "allele" refers to one of the variant forms of a biallelic or multiallelic alteration, differing from other forms in its nucleotide sequence. Typically the first identified allele is designated as the original allele whereas other alleles are designated as alternative alleles. Diploid organisms may be homozygous or heterozygous for an allelic form.
The term "polymorphism" as used herein refers to the occurrence of two or more alternative genomic sequences or alleles between or among different genomes or individuals. "Polymorphic" refers to the condition in which two or more variants of a specific genomic sequence can be found in a population. A "polymorphic site" is the locus at which the variation occurs. A polymorphism may comprise a substitution, deletion or insertion of one or more nucleotides. A single nucleotide polymorphism is a single base pair change. Typically a single nucleotide polymorphism is the replacement of one nucleotide by another nucleotide at the polymorphic site. A "single nucleotide polymorphism" (SNP) refers to a sequence polymorphism differing in a single base pair.
The term "antibody" as used herein encompasses monoclonal and polyclonal antibodies, chimeric, humanized, fully human, bispecific or multispecific antibodies as well as fragments thereof such as single chain antibodies (scFv) or domain antibodies, as further explained below.
Within the context of this invention, the term "selective" binding indicates that the antibodies preferentially bind the target polypeptide or epitope, i.e., with a higher affinity than any binding to any other antigen or epitope. In other words, binding to the target polypeptide can be discriminated from non-specific binding to other antigens. Antibodies of this invention may be monoclonal or polyclonal antibodies, or fragments or derivative thereof having substantially the same antigen specificity. The term fragment includes any binding portion of an antibody, such as Fab, F(ab')2, CDR domains, etc. Derivatives include human or humanized antibodies, polyfunctional antibodies, single-chain antibodies (e.g., ScFv), etc. Methods for producing antibodies, fragments or derivatives thereof are well known in the art, including immunization of an animal and collection of serum (polyclonal) or spleen cells (to produce hybridomas by fusion with appropriate cell lines).
Methods of producing polyclonal antibodies from various species, including rodents, primates and horses, have been described for instance in Vaitukaitis et al. (J Clin Endocrinol Metab. 33 (1971) p. 988). Briefly, the antigen is combined with an adjuvant (e.g., Freund's adjuvant) and administered to an animal, typically by sub-cutaneous injection. Repeated injections may be performed. Blood samples are collected and immunoglobulins or serum are separared. Methods of producing monoclonal antibodies may be found, for instance, in
Harlow et al (Antibodies: A laboratory Manual, CSH Press, 1988) or in Kohler et al (Nature 256 (1975) 495), incorporated therein by reference. Briefly, these methods comprise immunizing an animal with the antigen, subsequently recovering spleen cells and fusing these cells with immortalized cells, such as myeloma cells, to produce hybridomas. Hybrodimas producing the desired monoclonal antibodies can be selected by limit dilutions. Antibodies may also be produced by selection of combinatorial libraries of immunoglobulins, as disclosed for instance in Ward et al (Nature 341 (1989) 544).
The antibodies may be coupled to heterologous moieties, such as toxins, labels, drugs or other therapeutic agents, covalently or not, either directly or through the use of coupling agents or linkers.
DETECTION AND DIAGNOSIS
The present invention provides novel means and methodologies for detecting or diagnosing multiple sclerosis, systemic lupus erythematosus, psoriasis and related disorders in a human subject. The present methods may be implemented at various development stages of said pathologies, including early, pre-symptomatic stages, and late stages, in adults, children and pre-birth. Furthermore, the invention is suited to determine the prognosis, to assess a predisposition to or a risk of development of pathology, to characterize the status of a disease or to define the most appropriate treatment regimen for a patient.
A particular object of this invention resides in a method of detecting the presence of or predisposition to multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in a subject, the method comprising detecting the presence of an alteration in a NRGl gene or polypeptide in a sample from the subject, the presence of such an alteration being indicative of the presence of or predisposition to multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in said subject. Another object of this invention relates to methods of assessing the response of a subject to a treatment of multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder, the methods comprising detecting the presence of an alteration in a NRGl gene or polypeptide in a sample from the subject, the presence of such an alteration being indicative of a responder subject.
Current medications for MS which are disease modifying treatments, i.e. modifying the course of MS, modulate or suppress the immune system. Medications for MS within the meaning of the present invention include the four FDA approved immunomodulating agents for RRMS: three beta interferons (Betaseron®, Berlex; Avonex®, Biogen; Rebif®, Serono) and Glatimarer Acetate (Copaxone®, Amgen). Medications for MS within the meaning of the present invention also include the FDA approved immunosuppressing drug for worsening MS, Mitoxantrone (Novantrone®, Amgen).
The terms "interferon (IFN)" and "interferon-beta (IFN-beta)", as used herein, are intended to include fibroblast interferon in particular of human origin, as obtained by isolation from biological fluids or as obtained by DNA recombinant techniques from prokaryotic or eukaryotic host cells, as well as its salts, functional derivatives, variants, analogs and active fragments.
IFN-beta suitable in accordance with the present invention is commercially available e.g. as Rebif® (Serono), Avonex® (Biogen) or Betaferon® (Schering). The use of interferons of human origin is also preferred in accordance with the present invention. The term interferon, as used herein, is intended to encompass salts, functional derivatives, variants, analogs and active fragments thereof.
Rebif® (recombinant human interferon-) is the latest development in interferon therapy for multiple sclerosis (MS) and represents a significant advance in treatment. Rebif® is interferon (IFN)-beta Ia, produced from mammalian cell lines. It was established that interferon beta- Ia given subcutaneously three times per week is efficacious in the treatment of Relapsing-Remitting Multiple Sclerosis (RRMS). Interferon beta- Ia can have a positive effect on the long-term course of MS by reducing number and severity of relapses and reducing the burden of the disease and disease activity as measured by MRI.
The dosing of IFN-β in the treatment of relapsing-remitting MS according to the invention depends on the type of IFN-β used.
In accordance with the present invention, where IFN is recombinant IFN-β Ib produced in E. CoIi, commercially available under the trademark Betaseron, it may preferably be administered sub-cutaneously every second day at a dosage of about of 250 to 300 g or 8 MIU to 9.6 MIU per person.
In accordance with the present invention, where IFN is recombinant IFN-β Ia, produced in Chinese Hamster Ovary cells (CHO cells), commercially available under the trademark Avonex, it may preferably be administered intra-muscularly once a week at a dosage of about of 3Og to 33 g or 6 MIU to 6.6 MIU per person.
In accordance with the present invention, when IFN is recombinant IFN-β Ia, produced in Chinese Hamster Ovary cells (CHO cells), commercially available under the trademark Rebif, it may preferably be administered sub-cutaneously three times a week (TIW) at a dosage of 22 to 44 g or 6 MIU to 12 MIU per person.
There is currently no cure for SLE and complete sustained remissions are rare. Although survival rates have improved, over one half of patients with systemic lupus erythematosus have permanent damage in one or more organ systems. Arthritis and cutaneous manifestations are most common, but renal, hematologic and neurologic manifestations contribute largely to morbidity and mortality. Treatment approaches emphasize using a combination of drugs to minimize chronic exposure to corticosteroids. Therapeutic choices depend on:
1. whether disease manifestations are life threatening or likely to cause organ damage, justifying aggressive therapies
2. whether manifestations are potentially reversible
3. the best approaches to preventing complications of diseases and its treatments Over 90 percent of patients with systemic lupus erythematosus eventually have a cutaneous manifestation of the disease, including malar rash, discoid lupus erythematosus, alopecia or aphthous stomatitis. The usual therapy for cutaneous lupus erythematosus is strict use of sun block, judicious use of topical steroids and antimalarial therapy. Some patients with very severe cases of discoid lupus erythematosus may not respond adequately to the usual dosage of hydroxychloroquine, which is 400 mg per day for a normal-sized adult. Quinacrine, in a dosage of 100 mg per day, can be added without increasing the risk of retinopathy, or the patient can be switched to chloroquine HCl (Aralen), in a dosage of 250 mg per day. At some point, over 90 percent of patients with systemic lupus erythematosus have polyarthralgias or polyarthritis because of the disease. Nonsteroidal anti- inflammatory drugs (NSAIDs) remain the mainstay of treatment in these patients, especially those who have mild polyarthralgias or polyarthritis/ It is preferable to avoid the more gastrotoxic NSAIDs, because patients with systemic lupus erythematosus may require NSAID treatment for years and may use NSAIDs in conjunction with other drugs such as corticosteroids, which increases the risk of gastric injury. In addition, NSAIDs may adversely affect renal iunction, a special concern because 50 percent of patients with systemic lupus erythematosus develop associated nephritis. One of the major worries for physicians and patients with systemic lupus erythematosus is lupus nephritis.
Not all lupus nephritis is severe. Patients with milder forms, including mesangial glomerulonephritis and focal proliferative glomerulonephritis, may respond to corticosteroid therapy alone or with steroid-sparing drugs such as azathioprine. Cyclophosphamide (Cytoxan) is more effective than corticosteroids alone for the treatment of severe forms of lupus nephritis (diffuse proliferative glomerulonephritis). One of the major complications of systemic lupus erythematosus is premature or accelerated atherosclerosis. Corticosteroid treatment increases the levels of cardiovascular risk factors, including weight, blood pressure, cholesterol and homocysteine levels. Patients with systemic lupus erythematosus who have already had a manifestation of antiphospholipid antibody syndrome require treatment. Patients who have had venous or arterial thrombosis appear to benefit from maintenance therapy with high- intensity (International Normalized Ratio of 3 to 4) warfarin (Coumadin). The most frequent hematologic manifestation of systemic lupus erythematosus is anemia, usually normochromic normocystic, reflecting chronic illness. Anemia in patients with systemic lupus erythematosus is most often associated with chronic disease or is related to iron deficiency. Classic autoimmune hemolytic anemia can present acutely (and severely) or as a chronic condition. Severe hemolytic anemia is treated initially with intravenous methylprednisolone, 1,000 mg per day for three days.
Treatment approaches for psoriasis depend on the severity of the symptoms. In mild forms of psoriasis, topical treatments are applied, such as vitamin D3 analogs, corticosteroids, retinoids and coal tar. Oily bath solutions and moisturizers have a soothing effect on the skin.
Moderate to severe psoriasis is usually treated with light therapy. This includes therapy by sunlight or Ultraviolet B. Also available are combination therapies combining oral or topical administration of Psoralen with exposure to Ultraviolet A. Light therapy may also be combined with administration of retinoids.
For more severe forms of psoriasis, usually systemic treatment by methotrexate, oral retinoids or cyclosporin is prescribed.
Recently, biologies, which interfere with the immune system, have been introduced as new psoriasis therapeuticals. For example, efalizumab (marketed under the tradename Raptiva) is a humanised monoclonal antibody, which is recombinantly produced in mammalian cells. It inhibits activation of T cells and reduces inflammation in psoriasis.
As will be discussed below in more details, the alteration ("susceptibility alteration") in a NRGl gene or polypeptide may be any nucleotide or amino acid alteration associated to multiple sclerosis, systemic lupus erythematosus or a related disease.
A susceptibility alteration in the NRGl gene may be any form of mutation(s), deletion(s), rearrangement(s) and/or insertion(s) in the coding and/or non-coding region of the gene, either isolated or in various combination(s). Mutations more specifically include point mutations. Deletions may encompass any region of two or more residues in a coding or non-coding portion of the gene. Typical deletions affect small regions, such as domains (introns) or repeated sequences or fragments of less than about 50 consecutive base pairs, although larger deletions may occur as well. Insertions may encompass the addition of one or several residues in a coding or non-coding portion of the gene. Insertions may typically comprise an addition of between 1 and 50 base pairs in the gene. Rearrangements include for instance sequence inversions. An alteration in the NRGl gene may also be an aberrant modification of the polynucleotide sequence, such as of the methylation pattern of the genomic DNA, allelic loss of the gene or allelic gain of the gene. The alteration may be silent (i.e., create no modification in the amino acid sequence of the protein), or may result, for instance, in amino acid substitutions, frameshift mutations, stop codons, RNA splicing, e.g. the presence of a non-wild type splicing pattern of a messenger RNA transcript, or RNA or protein instability or a non-wild type level of the NRGl polypeptide. Also, the alteration may result in the production of a polypeptide with altered function or stability, or cause a reduction or increase in protein expression levels.
Typical susceptibility alterations are single nucleotide polymorphisms (SNPs).
In this regard, the present invention now discloses several susceptibility alterations in the
NRGl gene, which are associated with multiple sclerosis, systemic lupus erythematosus or psoriasis. These mutations are reported in table 2 (tables 2a, 2b, 2c, 2d, 2e). In summary, the association results of the single biallelic alteration frequency analysis show that the NRGl gene is associated with multiple sclerosis, systemic lupus erythematosus and psoriasis.
Preferred genetic alterations linked to MS are disclosed in table 2a below.
Preferred genetic alterations linked to SLE are disclosed in table 2b below. Preferred genetic alterations linked to psoriasis are disclosed in table 2c below.
Most preferred alterations linked to SLE are disclosed in table 2d below. Most preferred alterations linked to psoriasis are disclosed in table 2e below.
SNPs reported in tables 2a, 2b and 2c show a significant (p<=0.05) allele or genotype frequency difference between cases and controls, or show a significant Hardy- Weinberg disequilibrium in cases (p<=0.02). In other words, SNPs were selected if, for at least one of the study populations: - the allelic test p value is <=0.05 or
- the genotypic test p value is <=0.05 or
- the Hardy- Weinberg test p-value in cases is <=0.02.
The most preferred alterations linked to SLE or psoriasis, reported in tables 2d and 2e, are those SNP for which the Mantel-Haenszel statistical test was significant. This means that the allele frequency difference between cases and controls is significant for these SNPs when the two SLE or psoriasis sets of data are analysed simultaneously and stratified by study populations. For MS, the Mantel-Haenszel statistical test was not significant for any of the SNPs; this means, that different SNPs are linked to MS in the different populations.
The chromosomal SNP positions ("pos") given in the present patent application are based on NCBI human genome Build 35. These position show associated alleles which are preferred markers of the invention. The sites of the SNPs ("site") are further identified by unique Affymetrix SNP identifiers referring to the Affymetrix genechip human mapping IOOK set. These Affymetrix identifiers refer to unique oligonucleotides of 25bp length which allow for an identification of the respective SNP independent of the sequence of a specific genome build/version.
Table 2a: Univariate results for MS collections
Legend for abbreviations in tables 2a, 2b and 2c
Only SNPs showing significant Pall (<0.05), Pgeno (<0.05) or Phwca (<0.02) are included in the univariate results tables.
Collection Name of the study collection
SNP_name Affymetrix SNP name
DNA strand -1 : reverse strand, +1 : forward strand
Position in gene Base 1 = base number 119199117 of chromosome 3 to base number 117011800
Alleles
Chr Chromosome
Position SNP position on chromosome in base pair, NCBI release 35
Ch. Allele The Chosen Allele is the allele which frequency is increased within cases as compared to controls K)
MAFctl Minor Allele frequency in control individuals -4
AFD Allele frequency difference between cases and controls
Pall: Allelic frequency test P-value between cases and controls
OR Odds Ratio for the 2x2 contingency table (Allele frequency comparison)
Pgeno: Genotypic frequency test P-value between cases and controls
Phwca: Hardy- Weinberg Equilibrium test P-value in cases.
Table 2a (1): Univariate Results for MS RENNES Collection
Table 2a (2): Univariate Results for MS HUDDINGE Collection
Table 2a (31: Univariate Results for MS SERACARE Collection
K)
Table 2b; univariate results for SLE collections
Table 2b (1): Univariate Results for SLE SWEDEN Collection
K)
5 Table 2b (2): Univariate Results for SLE ARGENTINA Collection
30
Table 2c: univariate results for psoriasis collections
Table 2c (1): Univariate Results for Psoriasis NICE Collection
O
31 Table 2c (2): Univariate Results for Psoriasis SERACARE Collection
W
Table 2d: MH results for SLE Collections
5 Legend for abbreviations in table 2d and 2e:
Are included in Mantel-Haenszel results tables the SNPs that:
1) Passed the quality control criteria in analysed collections (by disease) and;
10 2) Show a valid MH test (HT_P>0.05) and;
3) Show a significant P MH (<0.05).
Index
MH_SLE_ASw Mantel-Ηaenszel (MH) test has been performed with SNP data from the two SLE collections; Argentina (A) and Sweden
(Sw).
15 MH PSO NS Mantel-Haenszel (MH) test has been performed with SNP data from the two PSO collections; Nice(N) and Seracare (S). K) SNP name Affymetrix SNP name DNA strand Position in gene Alleles
20 Chr Chromosome
Position SNP position on chromosome in base pair HT_chi2 Heterogeneity test χ2 statistics HT_pvalue Heterogeneity test P-value OR Mantel-Haenszel Odds Ratio
25 OR CIl Mantel-Haenszel Odds Ratio confidence interval (inferior limit) OR CI2 Mantel-Haenszel Odds Ratio confidence interval (superior limit) Chi2 Mantel-Haenszel χ2 statistics P MH Mantel-Haenszel test P-value PaIlMSJL Allelic frequency test P-value for MS Rennes
30 Pall MS Ji Allelic frequency test P-value for MS Huddinge PaIlMS S Allelic frequency test P-value for MS Seracare
Pall SLE A Allelic frequency test P-value for SLE Argentine PaIl SLE Sw Allelic frequency test P-value for SLE Sweden
Table 2e: MH results for Psoriasis Collections
10 w w
A preferred embodiment of the present invention comprises the detection of the presence of a susceptibility alteration as disclosed in Tables 2a, 2b or 2c in the NRGl gene or RNA sequence of a subject, more particularly the detection of at least one alteration as disclosed in Table 2d or 2e, or any combination thereof.
A preferred object of this invention is a method of detecting the presence of or predisposition to multiple sclerosis or a related disorder in a subject, the method comprising detecting the presence or absence of the associated susceptibility alteration according to table 2a in a sample from the subject, the presence of the associated susceptibility alteration being indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject.
Another preferred object of this invention is a method of detecting the presence of or predisposition to systemic lupus erythematosus or a related disorder in a subject, the method comprising detecting the presence or absence of the associated susceptibility alteration according to table 2b or 2d in a sample from the subject, the presence of the associated susceptibility alteration being indicative of the presence of or predisposition to systemic lupus erythematosus or a related disorder in said subject.
Another preferred object of this invention is a method of detecting the presence of or predisposition to psoriasis or a related disorder in a subject, the method comprising detecting the presence or absence of the associated susceptibility alteration according to table 2c or 2e in a sample from the subject, the presence of the associated susceptibility alteration being indicative of the presence of or predisposition to psoriasis or a related disorder in said subject.
Now that the inventors have established the association between NRGl and multiple sclerosis, systemic lupus erythematosus or related diseases, it should be understood that additional susceptibility alterations can be identified within said gene or polypeptide, e.g., following the methodology disclosed in the examples. The presence of an alteration in the NRGl gene may be detected by any technique known per se to the skilled artisan (reviewed by Kwok et al., 2003), including sequencing, pyrosequencing, selective hybridisation, selective amplification and/or mass spectrometry including matrix-assisted laser desorption/ionization time-of- flight mass spectrometry (MALDI-TOF MS) (Gut et al., 2004). In a particular embodiment, the alteration is detected by selective nucleic acid amplification using one or several specific primers. The alteration is detected by selective hybridization using one or several specific probes.
Further techniques include gel electrophoresis-based genotyping methods such as PCR coupled with restriction fragment length polymorphism analysis, multiplex PCR, oligonucleotide ligation assay, and minisequencing; fluorescent dye-based genotyping technologies such as oligonucleotide ligation assay, pyrosequencing, single-base extension with fluorescence detection, homogeneous solution hybridization such as TaqMan, and molecular beacon genotyping; rolling circle amplification and Invader assays as well as DNA chip-based microarray and mass spectrometry genotyping technologies (Shi et al., 2001).
Furthermore, RNA expression of altered genes can be quantified by methods known in the art such as subtractive hybridisation, quantitative PCR, TaqMan, differential display reverse transcription PCR, serial, partial sequencing of cDNAs (sequencing of expressed sequenced tags (ESTs) and serial analysis of gene expression (SAGE)), or parallel hybridization of labeled cDNAs to specific probes immobilized on a grid (macro- and microarrays and DNA chips. Particular methods include allele-specific oligonucleotide (ASO), allele-specific amplification, fluorescent in situ hybridization (FISH) Southern and Northern blot, and clamped denaturing gel electrophoresis.
Protein expression analysis methods are known in the art and include 2-dimensional gel- electrophoresis, mass spectrometry and antibody microarrays (Freeman et al., 2004 and Zhu et al., 2003).
Sequencing can be carried out using techniques well known in the art, using automatic sequencers. The sequencing may be performed on the complete gene or, more preferably, on specific domains thereof, typically those known or suspected to carry deleterious mutations or other alterations.
Amplification may be performed according to various techniques known in the art, such as by polymerase chain reaction (PCR), ligase chain reaction (LCR) and strand displacement amplification (SDA). These techniques can be performed using commercially available reagents and protocols. A preferred technique is allele-specific PCR.
Nucleic acid primers useful for amplifying sequences from the NRGl gene are able to specifically hybridize with a portion of the NRGl gene that either flanks or overlaps with a susceptibility alteration. The primer sequence overlaps with the alteration when said alteration is contained within the sequence of the NRGl gene to which the primer hybridizes. The primer sequence flanks the alteration when the primer hybridizes with a portion of the NRGl gene that is preferably located at a distance below 300 bp of said alteration, even more preferably below 250, 200, 150, 100, 50, 40, 30 or 20 bp from said alteration. Preferably, the primer hybridizes with a portion of the NRGl gene that is at 5, 4, 3, 2, 1 bp distance or immediately adjacent to said alteration.
The invention also relates to the use of a nucleic acid primer or a pair of nucleic acid primers as described above in a method of detecting the presence of or predisposition to multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in a subject or in a method of assessing the response of a subject to a treatment of multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder.
According to another embodiment of the present invention, the methods involve the use of a nucleic acid probe specific for a NRGl or altered NRGl gene, followed by the detection of the presence of a hybrid. The probe may be used in suspension or immobilized on a substrate or support. The probe is typically labelled to facilitate detection of hybrids.
In this respect, a specific object of this invention is a nucleic acid probe complementary to and specific for a region of a NRGl gene that carries an alteration as described in Table 2a, 2b, 2c, 2d or 2e. The probes of the present invention are, more preferably, capable of discriminating between an altered and non-altered NRGl gene sequence, i.e., they specifically hybridise to a NRGl gene carrying a particular alteration as described above, and essentially do not hybridise under the same hybridization conditions or with the same stability to a NRGl gene lacking said alteration.
The invention also concerns the use of a nucleic acid probe as described above in a method of detecting the presence of or predisposition to multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in a subject or in a method of assessing the response of a subject to a treatment of multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder.
The detection methods can be performed in vitro, ex vivo or in vivo, preferably in vitro or ex vivo. They are typically performed on a sample from the subject, such as any biological sample containing nucleic acids or polypeptides. Examples of such samples include fluids, tissues, cell samples, organs, biopsies, etc. Most preferred samples are blood, plasma, saliva, urine, seminal fluid, etc. The sample may be collected according to conventional techniques and used directly for diagnosis or stored. In particular, they may be obtained by non-invasive methods, such as from tissue collections. The sample may be treated prior to performing the method, in order to render or improve availability of nucleic acids or polypeptides for testing. Treatments include, for instance, lysis (e.g., mechanical, physical, chemical, etc.), centrifugation, etc. Also, the nucleic acids and/or polypeptides may be pre- purified or enriched by conventional techniques, and/or reduced in complexity. Nucleic acids and polypeptides may also be treated with enzymes or other chemical or physical treatments to produce fragments thereof. Considering the high sensitivity of the claimed methods, very few amounts of sample are sufficient to perform the assay.
The sample is typically contacted with probes or primers as disclosed above. Such contacting may be performed in any suitable device, such as a plate, tube, well, glass, etc. The contacting may be performed on a substrate coated with said specific reagents, such as a nucleic acid array. The substrate may be a solid or semi-solid substrate such as any support comprising glass, plastic, nylon, paper, metal, polymers and the like. The substrate may be of various forms and sizes, such as a slide, a membrane, a bead, a column, a gel, etc. The contacting may be made under any condition suitable for a complex to be formed between the reagent and the nucleic acids of the sample. The finding of an altered NRGl gene or polypeptide in the sample is indicative of the presence, predisposition or stage of progression of multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in the subject. Typically, one only of the above-disclosed susceptibility alterations is assessed, or several of them, in combination(s).
In another aspect of the invention there is provided a kit for the identification of a genetic polymorphism pattern at the NRGl gene associated with increased risk of the presence of or predisposition to multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in a subject, said kit comprising:
(a) DNA sample collecting means, and
(b) means for determining a genetic polymorphism pattern for the NRGl gene.
DRUG SCREENING
As indicated above, the present invention also provides novel targets and methods for the screening of drug candidates or leads. These screening methods include binding assays and/or functional assays, and may be performed in vitro, in cell systems or in animals.
In this regard, a particular object of this invention resides in the use of a NRGl polypeptide as a target for screening candidate drugs for treating or preventing multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder.
Another object of this invention resides in methods of selecting biologically active compounds, said methods comprising contacting a candidate compound with a NRGl gene or polypeptide, and selecting compounds that bind said gene or polypeptide.
A further other object of this invention resides in methods of selecting biologically active compounds, said method comprising contacting a candidate compound with recombinant host cell expressing a NRGl polypeptide with a candidate compound, and selecting compounds that bind said NRGl polypeptide and/or that modulate the activity of the NRGl polypeptide. A "biologically active" compound denotes any compound having biological activity in a subject, preferably therapeutic activity, and further preferably a compound that can be used for treating multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder, or as a lead to develop drugs for treating multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder. A "biologically active" compound preferably is a compound that modulates the activity of NRGl.
The above methods may be conducted in vitro, using various devices and conditions, including with immobilized reagents, and may further comprise an additional step of assaying the activity of the selected compounds in a model of multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder, such as an animal model.
Binding to a target gene or polypeptide provides an indication as to the ability of the compound to modulate the activity of said target, and thus to affect a pathway leading to multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in a subject. The determination of binding may be performed by various techniques, such as by labelling of the candidate compound, by competition with a labelled reference ligand, etc. For in vitro binding assays, the polypeptides may be used in essentially pure form, in suspension, immobilized on a support, or expressed in a membrane (intact cell, membrane preparation, liposome, etc.).
Modulation of activity includes, without limitation, stimulation of the surface expression of the NRGl receptor, modulation of multimerization of said receptor (e.g., the formation of multimeric complexes with other sub-units), etc. The cells used in the assays may be any recombinant cell (i.e., any cell comprising a recombinant nucleic acid encoding a NRGl polypeptide) or any cell that expresses an endogenous NRGl polypeptide. Examples of such cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E.coli, Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established mammalian cell cultures (e.g., produced from fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.). Preferred selected compounds are agonists of NRGl, i.e., compounds that can bind to NRGl receptor and mimic the activity of an endogenous ligand thereof.
In a particular embodiment, the screening assays of the present invention use, either alone or in addition to another NRGl sequence, an altered NRGl gene or polypeptide, particularly a NRGl gene or polypeptide having an alteration as listed in Table 2a, 2b, 2c, 2d or 2e.
A further object of this invention resides in a method of selecting biologically active compounds, said method comprising contacting in vitro a test compound with a NRGl polypeptide according to the present invention and determining the ability of said test compound to modulate the activity of said NRGl polypeptide.
A further object of this invention resides in a method of selecting biologically active compounds, said method comprising contacting in vitro a test compound with a NRGl gene according to the present invention and determining the ability of said test compound to modulate the expression of said NRGl gene, preferably to stimulate expression thereof.
In another embodiment, this invention relates to a method of screening, selecting or identifying active compounds, particularly compounds active on multiple sclerosis, systemic lupus erythematosus, psoriasis or related disorders, the method comprising contacting a test compound with a recombinant host cell comprising a reporter construct, said reporter construct comprising a reporter gene under the control of a NRGl gene promoter, and selecting the test compounds that modulate (e.g. stimulate or reduce, preferably stimulate) expression of the reporter gene.
In another embodiment, this invention relates to the use of a NRGl polypeptide or fragment thereof, whereby the fragment is preferably a NRGl gene-specific fragment, for isolating or generating an agonist or stimulator of the NRGl polypeptide for the treatment of multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder, wherein said agonist or stimulator is selected from the group consisting of: 1. a specific antibody or fragment thereof including a) a chimeric, b) a humanized or c) a fully human antibody as well as
2. a bispecific or multispecific antibody,
3. a single chain (e.g. scFv) or
4. single domain antibody, or 5. a peptide- or non-peptide mimetic derived from said antibodies or
6. an antibody-mimetic such as a) an anticalin or b) a fibronectin-based binding molecule (e.g. trinectin or adnectin).
7. A natural ligand of NRGl .
The generation of peptide- or non-peptide mimetics from antibodies is known in the art (Saragovi et al., 1991 and Saragovi et al., 1992).
Anticalins are also known in the art (Vogt et al., 2004). Fibronectin-based binding molecules are described in US6818418 and WO2004029224. Furthermore, the test compound may be of various origin, nature and composition, such as any small molecule, nucleic acid, lipid, peptide, polypeptide including an antibody such as a chimeric, humanized or fully human antibody or an antibody fragment, peptide- or non- peptide mimetic derived therefrom as well as a bispecific or multispecific antibody, a single chain (e.g. scFv) or single domain antibody or an antibody-mimetic such as an anticalin or fibronectin-based binding molecule (e.g. trinectin or adnectin), etc., in isolated form or in mixture or combinations.
PHARMACEUTICAL COMPOSITIONS AND THERAPY
The present invention now discloses novel approaches to the treatment of multiple sclerosis, systemic lupus erythematosus, psoriasis and related disorders by modulating the activity or expression of a NRGl gene or polypeptide. More particularly, the present invention provides the first evidence of a correlation between said gene and said diseases in human subjects, and allows the design of novel therapeutic approaches based on a modulation, preferably a stimulation or increase of a NRGl activity. In this regard, a particular object of this invention resides in the use of a NRGl polypeptide, or a nucleic acid encoding the same, for the manufacture of a pharmaceutical composition for treating or preventing multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in a subject.
A preferred embodiment resides in the use of a NRGl polypeptide of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID No. 10 or most preferably, SEQ ID NO. 9 for the manufacture of a pharmaceutical composition for treating or preventing multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in a subject.
A further object of this invention resides in the use of a modulator of NRGl for the manufacture of a pharmaceutical composition for treating or preventing multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in a subject. Most preferably, the modulator is an agonist or activator of a NRGl polypeptide.
In a preferred embodiment, the modulator is a NRGl fusion protein. A fusion protein of NRGl may e.g. comprise an immunoglobulin fusion, i.e. a fused protein comprising all or part of a NRGl protein, which is fused to all or a portion of an immunoglobulin. Methods for making immunoglobulin fusion proteins are well known in the art, such as the ones described in WO 01/03737, for example. The person skilled in the art will appreciate that the resulting fusion protein of the invention substantially retains the biological activity of NRGl, which can be measured in in vitro assays. The fusion may be direct, or via a short linker peptide which can be as short as 1 to 3 amino acid residues in length or longer, for example, 13 amino acid residues in length. Said linker may be a tripeptide of the sequence E-F-M (Glu-Phe-Met), for example, or for example a 13 -amino acid linker sequence comprising Glu-Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe- Met introduced between the NRGl sequence and the immunoglobulin sequence. The resulting fusion protein has improved properties, such as an extended residence time in body fluids (half-life), increased specific activity, increased expression level, or the purification of the fusion protein is facilitated.
In a preferred embodiment, NRGl is fused to the constant region of an Ig molecule, e.g. an Fc portion of an Immunoglobulin. Preferably, it is fused to heavy chain regions, like the CH2 and CH3 domains, optionally with the hinge region of human IgGl, for example. The Fc part may e.g. be mutated in order to prevent unwanted activities, such as complement binding, binding to Fc receptors, or the like. Other isoforms of Ig molecules are also suitable for the generation of fusion proteins according to the present invention, such as isoforms IgG2 or IgG4, or other Ig classes, like IgM or IgA, for example. Fusion proteins may be monomeric or multimeric, hetero- or homomultimeric.
Further fusion proteins of NRGl may be prepared by fusing domains isolated from other proteins allowing the formation or dimers, trimers, etc. Examples for protein sequences allowing the multimerization of the polypeptides of the Invention are domains isolated from proteins such as hCG (WO 97/30161), collagen X (WO 04/33486), C4BP (WO 04/20639), Erb proteins (WO 98/02540), or coiled coil peptides (WO 01/00814). In one embodiment the NRGl is fused to the C-terminal peptide of the beta-chain of hCG.
In a particularly preferred embodiment the modulator is a fusion protein between the Fc region of an antibody and a NRGl polypeptide defined by SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID No. 10 or most preferably, SEQ ID NO. 9.
In another embodiment, the NRGl peptide is PEGylated, i.e. it is chemically modified by the attachment of poly(ethyleneglycol) (PEG). The PEG may be linear or branched. It stabilizes the NRGl peptide, may increase the half-life and improve the bioactivity.
In another preferred embodiment, the agonist is a natural ligand of NRGl or NRGl receptor, or an antibody, such as a chimeric, humanized or fully human antibody or an antibody fragment, peptide- or non-peptide mimetic derived therefrom as well as a bispecific or multispecific antibody, a single chain (e.g. scFv) or single domain antibody or an antibody-mimetic such as an anticalin or fibronectin-based binding molecule (e.g. trinectin or adnectin), that selectively binds NRGl. Single domain antibodies, also called domain antibodies or dAbs, are the smallest functional binding units of antibodies, corresponding to the variable regions of either the heavy (VH) or light (VL) chains of human antibodies. Domain antibodies have a molecular weight of approximately 13 kDa, or less than one-tenth the size of a full antibody. In contrast to conventional antibodies, domain antibodies are well expressed in bacterial, yeast, and mammalian cell systems. In addition, many domain antibodies are highly stable and retain activity even after being subjected to harsh conditions, such as freeze-drying or heat denaturation which makes them amenable to a wide range of pharmaceutical formulation conditions and manufacture processes. In this regard, a further object of this invention is an antibody, or a fragment or derivative thereof, that selectively binds a NRGl polypeptide variant as disclosed above. In a more specific embodiment, the antibody, fragment or derivative thereof selectively binds NRGl polypeptide having the sequence as defined by SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 10 or most preferably, SEQ ID NO. 9.
In another embodiment, the modulator is an inhibitor or antagonist of a NRGl polypeptide.
A further object of this invention resides in a modulating antibody that selectively binds a NRGl polypeptide as disclosed above, in particular a NRGl polypeptide having the sequence as defined by SEQ ID No. 9. Modulating antibodies, which selectively bind NRGl polypeptide, have the effect of changing the biological activity of NRGl. Such modulating antibodies may be valuable in the treatment or prevention of MS, SLE and/or psoriasis.
A further object of this invention resides in a pharmaceutical composition comprising a functional NRGl polypeptide or a nucleic acid encoding a NRGl polypeptide or a vector encoding the same, and a pharmaceutically acceptable diluent or carrier.
A further object of this invention resides in a pharmaceutical composition comprising a modulator of NRGl as defined above, and a pharmaceutically acceptable diluent or carrier. The modulator may be an inhibitor or antagonist of NRGl or, most preferably, an activator or agonist of NRGl as defined above, including natural ligands.
The above uses or compositions are particularly suited for treating or preventing multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in a subject presenting an alteration in the NRGl gene or polypeptide, particularly in a subject presenting a SNP as described in Table 2a, 2b, 2c, 2d or 2e. To prepare a pharmaceutical composition according to the invention, a functional NRGl polypeptide or a nucleic acid encoding a NRGl polypeptide or a vector encoding the same or a NRGl modulator is intimately admixed with a pharmaceutical diluent or carrier according to conventional pharmaceutical compounding techniques known to the skilled practitioner. The NRGl modulator may be an antibody that selectively binds NRGl or a natural modulator of NRGl.
Suitable diluents and carriers may take a wide variety of forms depending on the desired route of administration. Pharmaceutical compositions according to the present invention can, for example, be administered orally or parenterally, i.e. intravenously, intramuscularly or subcutaneously, by inhalation or intranasally.
Pharmaceutical compositions suitable for oral administration can be solids or liquids and can, for example, be in the form of tablets, pills, dragees, gelatin capsules, solutions, syrups, chewing-gums and the like. To this end the active ingredient may be mixed with an inert diluent or a non-toxic pharmaceutically acceptable carrier.
The invention also contemplates compositions, which can release the active substance in a controlled manner. Pharmaceutical compositions, which can be used for parenteral administration, are in conventional form such as aqueous or oily solutions or suspensions generally contained in ampoules, disposable syringes, glass or plastic vials or infusion containers. In addition to the active ingredient, these solutions or suspensions can optionally also contain a sterile diluent such as water for injection, a physiological saline solution, oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents, antibacterial agents, and agents for adjusting the osmolarity.
These pharmaceutical forms are prepared using methods, which are routinely used by pharmacists.
The amount of active ingredient in the pharmaceutical compositions can fall within a wide range of concentrations and depends on a variety of factors such as the patient's sex, age, weight and medical condition, as well as on the method of administration. The daily dose can also fall within a wide range of dosage units. It should be understood that the specific doses can be adapted to particular cases depending on the individual requirements, at the physician's discretion. Another object of this invention is an isolated or recombinant NRGl gene or a fragment thereof, wherein said gene or fragment comprises a SNP selected from Table 2a, 2b, 2c, 2d or 2e.
The invention also relates to any vector comprising a nucleic acid as defined above. The vector may be any plasmid, phage, virus, episome, artificial chromosome, and the like. In a particular embodiment, the vector is a recombinant virus. Viral vectors may be produced from different types of viruses, including without limitation baculoviruses, retroviruses, adenoviruses, AAVs, etc., according to recombinant DNA techniques known in the art. The recombinant virus is typically replication-defective, even more preferably selected from El- and/or E4-defective adenoviruses, Gag-, pol- and/or env-defective retroviruses and Rep- and/or Cap-defective AAVs. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO95/14785, WO96/22378, US5,882,877, US6,013,516, US4.861.719, US5,278,056 and WO94/19478.
A further aspect of this invention is a recombinant host cell comprising a vector or a nucleic acid as defined above. The recombinant cell may be any prokaryotic or eukaryotic cells as discussed above. The recombinant cell preferably expresses a recombinant NRGl polypeptide at its surface.
A preferred embodiment of the invention is the use of an activator or agonist of NRGl in the preparation of a medicament for the treatment of multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder.
A particularly preferred embodiment of the invention is the use of an activator or agonist of NRGl in the preparation of a medicament for the treatment of multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder wherein the activator or agonist is an antibody such as a chimeric, humanized or fully human antibody or an antibody fragment, peptide- or non-peptide mimetic derived therefrom as well as a bispecific or multispecific antibody, a single chain (e.g. scFv) or single domain antibody or an antibody-mimetic such as an anticalin or fibronectin-based binding molecule (e.g. trinectin or adnectin).
The invention also relates to a method of treating or preventing multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in a subject, the method comprising administering to said subject a compound that modulates, preferably that activates or mimics, expression or activity of a NRGl gene or polypeptide as defined above.
A particular embodiment of the present invention resides in a method of treating or preventing multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in a subject, the method comprising (i) detecting in a sample from the subject the presence of an alteration in the NRGl gene or polypeptide as defined above and (ii) administering to said subject an agonist of NRGl . Preferably, said alteration is selected from the group consisting of an alteration as disclosed in Table 2a, 2b, 2c, 2d or 2e.
In another aspect of the invention there is provided a method of treating multiple sclerosis, or a related disorder in a subject in need of such treatment comprising of administering to the subject a compound that modulates the synthesis, expression or activity of one or more of the genes or gene products of the genes listed in Table 2a in a therapeutically effective amount so that at least one symptom of the multiple sclerosis or a related disorder is ameliorated.
In another aspect of the invention there is provided a method of treating systemic lupus erythematosus, or a related disorder in a subject in need of such treatment comprising of administering to the subject a compound that modulates the synthesis, expression or activity of one or more of the genes or gene products of the genes listed in Table 2b or Table 2d in a therapeutically effective amount so that at least one symptom of the systemic lupus erythematosus or a related disorder is ameliorated.
In another aspect of the invention there is provided a method of treating psoriasis, or a related disorder in a subject in need of such treatment comprising of administering to the subject a compound that modulates the synthesis, expression or activity of one or more of the genes or gene products of the genes listed in Table 2c or Table 2e in a therapeutically effective amount so that at least one symptom of the psoriasis or a related disorder is ameliorated.
In another aspect of the invention there is provided a method of treating multiple sclerosis or a related disorder in a subject in need of such treatment wherein the subject has a susceptibility alteration in a NRGl gene comprising of administering to the subject a therapeutically effective amount of a medication for MS, such as for example interferon- beta, preferably interferon-beta Ia.
Preferably the susceptibility alteration is selected from one or more of the susceptibility alterations listed in Table 2a. Preferably the susceptibility alteration is a single nucleotide polymorphism.
Further aspects and advantages of the present invention will be disclosed in the following section, which should be regarded as illustrative and not limiting the scope of the present application.
EXAMPLES
Example 1: Materials and Methods
1.1 Collections of patients and DNA banking - Subjects
1.1.1 Multiple Sclerosis (MS) samples The study comprised three collections of unrelated patients with MS, and unrelated healthy controls recruited from the neurological Department of Rennes (France: 314 cases; 353 controls), Huddinge (Sweden: 279 cases; 301 controls) hospitals and SeraCare (USA: 289 cases; 289 controls).
Table Ia provides a summary for the description and stratification study of the different collections.
Informed consent was given by each individual participating in the study, according to the Helsinki Convention (1964).
The following variables were recorded for each patient: sex, ethnic background, family history with regards to MS, diagnostic category, disease course, age at disease onset, results of cerebrospinal fluid (CSF) and Magnetic Resonance Imaging (MRI) examination, Expanded Disability Status scale (EDSS) score (Kurtzke, 1983) and disease duration at last inter-relapse clinical examination, using the latest data available
Disease courses were classified as relapsing-remitting (RR), relapsing-secondary progressive (SP), or primary-progressive (PP) as follows:
• RR: relapses with full recovery or with a residual deficit and lack of progression between relapses;
• SP: initial RR MS followed by progression;
• PP: Disease progression from onset
Selected for the MS study were patients with Primary progressive type, remitting-relapsing type or secondary progressive type MS, who have been diagnosed as MS according to the criteria of Mc Donald et al. (2001). Rennes collection:
Each patient and control subject included in the analysis had to be born in Bretagne, France as well as their parents and grand-parents.
The female / male ratio in the patient group was 2.14 (214 Females & 100 Males) with a mean age of 44 [19;68] years and in the control group 2.07 (238 Females & 115 Males) with a mean age of 35 [18;56] years.
Huddinge collection:
All participants in this study were drawn from a homogeneous population of Huddinge, Sweden.
The female / male ratio in the patient group was 2.4 (196 Females & 83 Males) with a mean age of 47 [22;75]. The control group in Huddinge collection included 301 (214 Females & 87 Males) healthy volunteers and the Female / male ratio was 2.5. Ages ranged from 22 to 73 years with a mean age of 47 years.
Seracare collection:
All the subjects included in the study were Caucasian from USA.
The group of cases included 289 subjects with a sex ratio of 5.7 (246 females and 43 males) and a mean age of 50 [32;74] years. The group of healthy volunteers included 289 individuals with a sex ratio of 5.7 (246 females and 43 males) and a mean age of 48.7 [36;75] years.
Table Ia: Description and stratification study of the different collections for MS
NS, Non significative P value 1.1.2 Systemic Lupus Erythematosus (SLE) samples
The study comprised two collections of unrelated patients with SLE, and unrelated healthy controls recruited from a multicenter study at Uppsala University: a Swedish collection (279 cases; 515 controls) and an Argentinean collection (255 cases; 256 controls).
Table Ib provides a summary for the description and stratification study of the different collections.
Informed consent was given by each individual participating in the study, according to the Helsinki Convention (1964).
The following variables were recorded for each patient: sex, birth, ethnic background, family history with regards to SLE, age at disease diagnosis and clinical manifestations according to the American College of Rheumatology (ACR) criteria (Hochberg 1997; Tan et al. 1982), using the latest data available.
Selected for the SLE study were individuals who fulfilled at least 4 of the 11 ACR criteria.
Swedish collection:
The mean age of the Swedish patients was 56 [23-89] years and 58 [13-89] years for females and males, respectively. The mean age of the Swedish controls was 44 [22-80] and 55 [45-73] for females and males, respectively. Females represent 89.2% (sex ratio = 8.3) and 88.3% (sex ration = 7.6) of individuals within the Swedish patient and control groups, respectively.
Argentinean collection:
The mean age of the Argentinean patients was 42 [16-75] years and 37 [23-50] years for females and males, respectively. The mean age of the Argentinean controls was 42 [17-77] and 37 [15-54] for females and males, respectively. Females represent 93.3% (sex ratio= 14) and 92.2% (11.8) of individuals within the Argentinean patient and control groups, respectively. It has to be noted that initially, the Swedish population had a big sex ratio difference between cases and controls. Therefore we added all control females from the Swedish sample collected for the MS study and removed the youngest controls males in order to restore comparable sex ratio between cases and controls. Table Ib: Description and stratification study of the different collections for SLE
No clinical subgroup has been studied yet for SLE and the age at disease onset is a variable very difficult to obtain with accuracy for SLE. Therefore these two variables are not shown. NS, Non significative P valmue
1.1.3 Psoriasis samples
The study comprised two collections of unrelated patients with Psoriasis, and unrelated healthy controls recruited from SeraCare (USA: 206 cases and 298 controls) and from Nice Hospital (Frannce: 376 cases and 402 controls).
Table Ic provides a summary for the description and stratification study of the different collections.
Informed consent was given by each individual participating in the study, according to the
Helsinki Convention (1964). The following variables were recorded for each patient: sex, ethnic background, age, age at onset, disease activity assessment as PASI (Psoriasis Area and Severity Index) or BSA (Body Surface Area).
5 SeraCare collection:
Each patient and control subject included in the analysis had Caucasian origin for at least two generations. The female/male ratio in the patient group was 0.97 (128 females & 132 males) with a mean age of 47.9 [20;72] years and in the control group 1.04 (152 females & 146 males) with a mean age of 47.8 [18;75] years.
10
Nice collection:
Each patient and control subject included in the analysis had Caucasian origin for at least two generations. The female/male ratio in the patient group was 0.95 (183 females & 193 males) with a mean age of 45.5 [18;75] years and in the control group 2.7 (294 females &
15 108 males) with a mean age of 36.65 [18;64] years.
Table Ic: Description and stratification of the different collections for Psoriasis
1.2. DNA extraction:
Genomic DNA was extracted from EDTA anticoagulated peripheral blood according to a standard proteinase K digestion and a modified salting out extraction method of Miller and co-workers (1988).
5 2.1 GENOTYPING
2.1.1 Methods for stratification analyses: Beckman UHT protocol:
Assay Design
Design of the two PCR primers and one SNP-IT primer for each alteration set was performed using Autoprimer.com (http://www.autoprimer.com). The Autoprimer.com 10 design engine reads each sequence and designs three primers; forward and reverse PCR primers and a SNP-IT primer for the single base extension step. Once primers are picked for each sequence, they are then assembled into groups of 12 by SNP extension type (e.g., A/G, T/C).
Each group, or panel of 12 alterations, must be of the same extension type for processing 15 on the UHT since each extension mix contains two labeled terminators (Bodipy-
Fluorescein and TAMRA). Each group of twelve is referred to as a panel of alterations. Autoprimer.com automatically optimizes the grouping of the alterations by extension mix and appends tag sequences to the 5' ends of the SNP-IT primers, which are complementary to the tags immobilized on the microarray plate.
20 PCR
A five-microliter PCR was performed in 384-well plates (MJ Research, Watertown, MA, USA) using 75-uM dNTPs and 0.5U AmpliTaq® Gold (Applied Biosystems) in IXPCR buffer. Two nanograms of genomic DNA were used in each reaction.
The 24 PCR primers were pooled and added such that each was at a final concentration of 25 50 nM. Thermal cycling was performed in DNA Engine Tetrad thermal cyclers (MJ
Research) using the following program: 95°C for 5 seconds followed by 45 cycles of 95°C for 30 seconds; 50°-55°C for 55 seconds; 72°C for 30 seconds. The first six cycles used an annealing temperature of 50°C after which the annealing temperature was increased by 0.2°C in the subsequent cycles until the annealing temperature reached 55°C. After the last cycle, the reaction was held at 72°C for 7 minutes followed by a 4°C hold.
PCR Clean-Up
Following PCR, 384-well plates were centrifuged briefly to collect the contents and 3 uL of a cocktail containing 0.67 U exonuclease I (USB, Cleveland, OH, USA) and 0.33 U shrimp alkaline phosphatase (SAP; USB) was added. Sealed plates were incubated for 30 minutes at 37°C to degrade residual PCR primers and dNTPs, and 10 minutes at 100°C to inactivate the enzymes.
SNP-IT Reaction
To the ExoI/SAP-treated PCR, we added 7 uL of a cocktail containing one TAMRA- labeled and one Bodipy-Fluorescein- labeled nucleotide terminator (PE-NEN, Boston, MA, and Molecular Probes, Eugene, OR, USA), the two remaining unlabeled terminators, 26.6 mM MgC12, 266 mM Tris-HCl pH 9.5, two allele- specific self-extension control primers, and a thermostable, 3'exonuclease-deficient polymerase such as Thermo Sequenase (Amersham Biosciences, Piscataway, NJ, USA). The total reaction volume was 15 uL. Plates were re-sealed and thermal cycled using the following program: 96°C for 3 minutes followed by 45 cycles of 94°C for 20 seconds; 40°C for 11 seconds. After the last cycle, the reaction was held at 4°C.
Hybridization and Washing
Following SNP-IT extension, 8 μL of hybridization buffer (5M NaCl, 0.5 M EDTA, 580 mM morpholinoethane sulphonic acid (MES) pH 6.6, IX Denhardt's Solution) was added and a portion of the mixture was applied to the well of a UHT microarray plate. Plates were incubated in a humidified container at 42°C for 2 hours to promote hybridization of the SNP-IT primers to their complementary immobilized tags. Plates were rinsed with UHT wash buffer using a conventional plate washer to remove unhybridized material and were then ready for imaging.
SNPstream UHT Array Imager The SNPstream Array Imager is based upon a two-laser, two-color approach. Each sample is illuminated with a 488-nm laser beam and subsequently with a 532-nm laser beam to excite the fluorescent oligonucleotides captured on the UHT microarray plates. The system contains two emission band filters. Fluorescence emission from 488-nm excitation (Bodipy- Fluorescein) is captured in a band 50 nm wide, centered at 535nm. Fluorescence emission from 532-nm excitation (TAMRA) is captured in a band 55 nm wide, centered at 590 nm. A colorcorrected custom lens, of high numerical aperture and 100-um A 2 X3 well area is imaged per frame. Sixty- four 2 X3 well images/color are taken per plate for a total of 384 wells. Total time required for the process is approximately seven minutes/plate.
Data Analysis
Generation of genotype calls from spots detected using the SNPstream UHT Array Imager involves two discrete steps. First, the location and intensity of a spot within the well and plate is determined for each wavelength; second, a genotype call is made based on the relative fluorescent intensities of each spot. Once a genotype call has been made, results are written to an Oracle® database where the data can easily be retrieved for viewing.
Spot detection is an automatic process performed by UHTImage software. Positive controls in each well are used to align the grids around the 4 X4 element array. Once a grid is drawn, each spot is analyzed for morphology (i.e., circular shape and regular pixel intensity across each spot). Spots with low intensity or unusual morphology are marked as empty or fail. For each spot that passes the morphology test, an intensity value is generated and loaded into the UHT database. Failed spots are carried through the analysis but are flagged for the user to review.
Genotype calling is performed once all spot intensities are in the database for each sample within a plate. Each SNP is analyzed separately using UHT GetGenqssoftware. This software automatically creates genotype calls based on the intensity value of each spot at each wavelength for a given sample. These calls are based on how the sample points cluster when plotted on a X, Y graph where X corresponds to the intensity in the 488-nm channel and Y to that of the 532-nm channel. If a point falls between clusters or the intensity of the point is too low, the sample is failed. Otherwise the point is called as XX, XY, or YY with the X's and Y's being replaced by the actual allele calls (A5C5G5T). UHT GetGenos uses a proprietary algorithm to determine the clusters and the genotypes for each sample. After the genotype calling, the results are stored in the database by microarray plate number, well, and spot location.
2.1.2 Methods for Whole Genome analysis: Affymetrix method:
Use of Affymetrix genotyping technology have been described in Kennedy, G.C., et al. Nature Biotechnology 21, 1233-1237, 2003 (Large-scale genotyping of complex DNA). ; Liu, W.M., et al. Bioinformatics 19, 2397-2403, 2003. (Algorithms for Large Scale Genotyping Microarrays) ;Matsuzaki, H., et al. Genome Research 3, 414-25, 2004. (Parallel Genotyping of over 10,000 SNPs using a One Primer Assay on a High Density Oligonucleotide Array) ;Paez, J.G., et al. Nucleic Acids Research 32(9), 2004. (Genome coverage and sequence fidelity of Φ29 polymerase-based multiple strand displacement whole genome amplification); Matsuzaki, H., et al.. Nature Methods, 1, 109-111, 2004 (Genotyping over 100,000 SNPs on a Pair of Oligonucleotide Arrays); Huang, J., et al., M.H. Human Genomics 1(4), 287-99, 2004. (Whole Genome DNA Copy Number Changes Identified by High Density Oligonucleotide Arrays); Mitra N, et al. Cancer Research 64 (21), 8116-25, 2004 (Localization of cancer susceptibility genes by genome-wide single- nucleotide polymorphism linkage-disequilibrium mapping), all of which are herewith incorporated by reference.
DNA preparation:
For each individual assayed, 250 ng of genomic DNA are digested separately with 10 U of Xbal or HmdIII (New England BioLabs) in volumes of 20 μL for 2 hours at 37 °C. Following heat inactivation at 70 °C for 20 minutes, 0.25 μM of Xbal adaptor (5'-ATT ATG AGC ACG ACA GAC GCC TGA TCT-3' and 5 'phosphate -CTA GAG ATC AGG CGT CTG TCG TGC TCA TAA- 3') (Affymetrix), or HmdIII adaptor (5'-ATT ATG AGC ACG ACA GAC GCC TGA TCT-3' and 5 'phosphate -AGC TAG ATC AGG CGT CTG TCG TGC TCA TAA-3') (Affymetrix) are ligated to the digested DNAs with T4 DNA Ligase (New England BioLabs) in 25 μL for 2 hours at 16 °C. The ligations are stopped by heating to 70 °C for 20 minutes, and then diluted 4- fold with water. For each ligation reaction, two to three PCRs are run in order to generate > 40 μg of PCR products. Each PCR contains 10 μL of the diluted ligation reactions (25 ng of starting DNA) in 100 μL volumes containing 1.0 μM of primer (5'-ATT ATG AGC ACG ACA GAC GCC TGA TCT-3'), 0.30 mM dNTPs, 1.0 mM MgSO4, 5 U Platinum® Pfa Polymerase (Invitrogen), PCR Enhancer (Invitrogen) and Pfx Amplification Buffer (Invitrogen). 30 cycles of PCRs are run with the following cycling program: 94 °C denaturation for 15 seconds, 60 °C annealing for 30 seconds, and 68 °C extension for 60 seconds. As a check, 3 μL of PCR products are visualized on 2% TBE agarose gels to confirm the size range of amplicons. The PCR products are purified over MinElute 96 UF PCR Purification plates (Qiagen), and recovered in 40 μL of EB buffer (Qiagen). PCR yields are measured by absorbance readings at 260 nm, and adjusted to a concentration of 40 μg per 45 μl. To allow efficient hybridization to 25-mer oligonucleotide probes, the PCR products are fragmented to < 100 bp with DNAse I. 0.20 U of DNAse I (Affymetrix) is added to 40 ug of purified PCR amplicons in a 55 μL volume containing Fragmentation Buffer (Affymetrix) for 35 minutes at 37 °C, followed by heat inactivation at 95 °C for 15 minutes. Fragmentation products are visualized on 4% TBE agarose gels. The 3' ends of the fragmented amplicons are biotinlyated by adding 214 μM of a proprietary DNA labeling reagent (Affymetrix) using Terminal Deoxynucleotidyl Transferase (Affymetrix) in 70 μL volumes for 2 hours at 37 °C, followed by heat inactivation at 95 °C for 15 minutes.
Allele Specific Hybridization to Oligonucleotide Arrays
The fragmented and biotinylated PCR amplicons are combined with 11.5 μg/mL human Cot-1 (Invitrogen) and 115 μg/mL herring sperm (Promega) DNAs. The DNAs are added to a hybridization solution containing 2.69 M tetramethylamonium chloride (TMACl), 5.77 mM EDTA, 56 mM MES, 5 % DMSO, 2.5 X Denhardt's solution, and 0.0115% Tween-20 in a final volume of 260 μL. The hybridization solution was heated to 95 °C for 10 minutes then placed on ice. After warming to 48 °C for 2 minutes, 200 μL of the hybridization solution is injected into cartridges housing the oligonucleotide arrays (Affymetrix GeneChip® IOOK Mapping Set: 50K Array Xba 240 and 50K Array Genotyping over 100,000 SNPs Hind 240). Hybridizations are carried out at 48 °C for 16 to 18 hours in a rotisserie rotating at 60 rpm. Following the overnight hybridization, the arrays are washed with 6X SSPE and 0.01% Tween-20 at 25 °C, then more stringently washed with 0.6X SSPE and 0.01% Tween-20 at 45 °C. Hybridization signals are generated in a three step signal amplification process: lOμg/mL streptavidin R-phycoerythrin (SAPE) conjugate (Molecular Probes) is added to the biotinylated targets hybridized to the oligonucleotide probes, and washed with 6X SSPE and 0.01% Tween-20 at 25 °C; followed by the addition of 5μg/mL biotinylated goat anti-streptavidin (Vector) to increase the effective number of biotin molecules on the target; and finally SAPE is added once again and washed extensively with 6X SSPE and 0.01% Tween-20 at 30 °C. The SAPE and antibody were added to arrays in 6X SSPE, IX Denhardt's solution and 0.01% Tween-20 at 25 °C for 10 minutes each. Following the final wash, the arrays are kept in Holding buffer (10OmM MES, IM [Na+], 0.01% Tween-20). The washing and staining procedures are run on Affymetrix fluidics stations. Arrays are scanned using GCS3000 scanners with
AutoLoaders (Affymetrix). Scan images are processed to get hybridization signal intensity values using GCOS 2.0 software (Affymetrix). The DM genotype calling algorithm is implemented in GenoTyping Tools (GTT) (Affymetrix) and GDAS 3.0 (Affymetrix) analysis software.
2.2 STATISTICAL ANALYSIS
Design for MS study: It was decided to analyze 2 different populations in parallel to minimize the risk of type I errors (false positives) due to the relatively limited sample size. In our case, the 2 populations have the same heuristic value and neither one represents an exploratory or a confirmation sample. Rather they represent 2 complementary views of the same analytical problem and only positive results that are cross-confirmed are retained as valid. The following paragraphs detail the statistics that we applied to perform our analyses. A third population (SeraCare) was studied to further confirm the results.
Design for SLE study: It was decided to analyze 2 different populations in parallel to minimize the risk of type I errors (false positives) due to the relatively limited sample size. In our case, the 2 populations have the same heuristic value and neither one represents an exploratory or a confirmation sample. Rather they represent 2 complementary views of the same analytical problem and only positive results that are cross-confirmed are retained as valid.
Design for psoriasis study: It was decided to analyze 2 different populations in parallel to minimize the risk of type I errors (false positives) due to the relatively limited sample size. In our case, the 2 populations have the same heuristic value and neither one represents an exploratory or a confirmation sample. Rather they represent 2 complementary views of the same analytical problem and only positive results that are cross-confirmed are retained as valid.
The following paragraphs detail the statistics that we applied to perform our analyses.
PART A: Descriptive statistics
2.2.1 Genetic homogeneity: FST test and Pritchard and Rosenberg test
A stratification effect is a non-homogeneous representation of populations between the case and the control groups due to genetic heterogeneity, which may lead to spurious association results and replication problems.
If cases and controls contain an admixture of different groups (for example, based on ethnicity), we expect to find a consistent pattern of allele-frequency differences between cases and controls, at many random loci throughout the genome, this difference exceeding the significant p-value for association at more than 5% of these random loci.
The power to detect stratification will depend on the number of loci used to test for homogeneity. Consequently, we have chosen a large number of unlinked SNPs (n=86). Stratification is always performed with the same set of SNP. These SNPs have been selected under the following conditions:
1 Minor allele frequency > 30% (highly polymorph)
2 Inter SNP distance > 10Mb (genetically independent)
3 Location in each chromosome (genome wide scale) but not in a known associated region for the studied disease (not associated with the disease)
All cases and controls were genotyped for all the unlinked genetic alterations set using the Beckman technology.
Two methods testing for genetic heterogeneity have been implemented in Serono Genetics Institute:
1. F st test (Wright 1951) is an ANOVA-based method. The Fst value quantifies the loss of heterozygosity due to existence of a hierarchical structure. If it is different from 0, it means that the population under study is genetically heterogeneous, since allelic frequencies are different between populations.
2. Pritchard & Rosenberg test (AmJ.Hum.Genet. 65:220-228, 1999) calculates an overall chi-square statistic of allelic frequency differences between cases and controls.
If the Fst and the Pritchard & Rosenberg tests do not show statistically significant results (p-value > 5%), cases and controls are considered homogenous and can be used for case- control association study.
However, statistically significant results at these tests do not necessarily mean that these populations must be discarded. Further analyses can assign each subject to a specific subpopulation and identify outliers (Structure software, Pritchard, 2002) that can be removed in order to restore homogeneity.
When the admixture is such that we can not identify clear subpopulations, we can adopt another approach, termed Genomic Control (Devlin andRoeder 1999): given that in the presence of population substructure, the standard chi-square statistic is inflated by a multiplicative factor, which is proportional to the degree of stratification, we can estimate and incorporate this multiplicative factor (lambda) into the disease - susceptibility alteration association tests (by rescaling the chi-square statistic) to correct for background population differences.
PART B: Inferential statistics
2.2.2 Univariate analysis
I. Hardy- Weinberg Equilibrium / Disequilibrium [HWE/D] : significance in Cases and in Controls
The Hardy- Weinberg law regulating equilibrium (HWE) is the central theory of population genetics, explaining why populations have a stable genetic pattern across generations and is based on four assumptions:
1. Populations are panmict (couples are formed at random) and their gametes meet randomly;
2. Populations are "Infinite" (large population size to minimize sampling variations); 3. There are no selection, mutation, migration (=no allele loss or allele gain);
4. Generations are discrete (no mating between different generations).
According to these hypotheses, the control population used in case-control association studies must respect this equilibrium, if sampled randomly. On the contrary, the population of cases can present some disequilibrium that may point to "mutations" underlying the disease, since cases are not a random representation of the general population.
Accordingly, we tested HWE for each SNP in the control population, and we removed from the study each SNP presenting a deviation from the equilibrium. In fact, any such deviation might be due to several different reasons, but especially to technical issues (e.g. neighbouring SNPs causing imbalance of the polymerase chain reaction products or affecting the genotyping assay). HWE test therefore serves two objectives: data review and quality check as well as detection of possible mutation.
The test described by Weir in Genetic Data Analysis II (Sinauer, 1996) has been implemented using a chi-square statistics (ldf). The SNPs with results showing significant deviation from HWE (pvalue < 0.02) were considered in disequilibrium and were not validated, a positive deviation demonstrating an excess of homozygotes (or lack of heterozygotes) and a negative deviation being due to an excess of heterozygotes (or lack of homozygotes).
Hardy- Weinberg equilibrium statistics were calculated separately for cases and controls data and Observed and Expected genotype frequencies were compared using a Pearson's χ2 test. A departure from Hardy- Weinberg equilibrium (HWE) in case population may indicate that a mutation had occurred, which could be responsible for increasing the risk for the disease.
II. Tests on allelic frequencies, gcnotypic frequencies, HWD In the univariate analysis (or Single Point Analysis), SNPs were analysed one by one. The Pearson's 2x2 χ2 test was used to compare allele frequencies between cases and controls, while we used a 3x2 χ2 test for the overall difference in genotype frequencies. The Exact Fisher test was performed wherever the minor expected frequency for each cell of the χ2 table is < 5.
Additional statistics include (i) the difference between allelic frequencies in cases and in controls (the larger the difference in allelic frequency for a given SNP, the more probable is an association between the genomic region containing that SNP and the disorder), (ii) the Odds Ratio (OR) of the association and (iii) the population Attributable Risk (pAR). The "chosen" allele is the allele for which the frequency is increased in cases compared to controls. Preferred single nucleotide polymorphisms indicative of multiple sclerosis, lupus erythematosus or psoriasis are the chosen alleles of Tables 2a, 2b, 2c, 2d and 2e.
In other words, if a single nucleotide polymorphism is the chosen allele defined in Table 2a, then the likelihood of the presence of or the predisposition to multiple sclerosis in a subject is increased. If a single nucleotide polymorphism is the chosen allele defined in Tables 2b or 2d, then the likelihood of the presence of or the predisposition to systemic lupus erythematosus in a subject is increased. If a single nucleotide polymorphism is the chosen allele defined in Tables 2c or 2e, then the likelihood of the presence of or the predisposition to psoriasis in a subject is increased.
We considered a p-value = or < 0.05 as threshold to consider the tests as significant for screening, with the only exception relative to HW test where the threshold is = or < 0.02.
III. Mantel Haenszel test: comparison of the significant findings across populations.
The relationships between genetic susceptibility to MS and allele frequencies have been studied for many SNPs (N = 95 938) in at least one of the two populations (Rennes & Huddinge). Data from most of these SNPs (N=82 925) are available for the two populations (Rennes & Huddinge): therefore, they represent the basis to evaluate associations that are observed in the two populations simultaneously. The relationships between genetic susceptibility to SLE and allele frequencies have been studied for many SNPs (N = 97 860) in at least one of the two populations (Argentine & Sweden). Data from most of these SNPs (N=81 697) are available for the two populations (Argentine & Sweden): therefore, they represent the basis to evaluate associations that are observed in the two populations simultaneously.
The relationships between genetic susceptibility to Psoriasis and allele frequencies have been studied for many SNPs (N=99610) in at least one of the two populations (SeraCare & Nice). Data from most of these SNPs (N=92463) are available for the two populations: therefore, they present the basis to evaluate associations that are observed in the two populations simultaneously.
We used the Mantel-Haenszel χ2 test which was designed for case-control studies in which the effect of an exposure-factor (Allele) on the outcome (MS, SLE or psoriasis) is investigated according to a stratification factor (Population).
A program was written at Serono Genetics Institute to perform the Mantel-Haenszel test using data from n independent populations (Principles of Biostatistics, Second Edition, Marcello Pagano & Kimberlee Gauvreau, Duxbury-Thomson Learning).
For the MS association study, none of the tested SNPs shows significant Mantel-Haenszel statistical test results. This means that the allele frequency difference between cases and controls is not significant for these SNPs when the three MS sets of data are analyzed simultaneously and stratified by study populations.
For SLE, three SNPs show significant Mantel-Haenszel test results. This means that the allele frequency difference between cases and controls is significant for these SNPs when the two SLE sets of data are analyzed simultaneously and stratified by study populations. This is explained by the fact that for these two SLE populations, the same trend is observed in both populations, i.e. the same allele is more common in cases than in controls and vice versa for the other allele.
For psoriasis, thirteen SNPs show significant Mantel-Haenszel test results. This means that the allele frequency difference between cases and controls is significant for these SNPs when the two psoriasis sets of data are analyzed simultaneously and stratified by study populations. This is explained by the fact that for these two psoriasis populations, the same trend is observed in both populations, i.e. the same allele is more common in cases than in controls and vice versa for the other allele.
2.2.3. Odds Ratio (OR)
By estimating the allelic Odds Ratio (OR) the probability of having the disease when carrying a given allele (= chosen [or 'risk'] allele) compared to not carrying it is evaluated. An OR higher than 1 shows that the probability of having multiple sclerosis or systemic lupus erythematosus respectively is higher when carrying the 'risk' allele [or genotype or haplotype] than when carrying the other ones.
The genotypic OR allows the identification of the 'risk' genotype(s) for an associated biallelic susceptibility alteration. The genotypic odds ratio was calculated and tables 2a, 2b and 2c show the significant results.
Example 2: Therapeutic treatment
The following test method can be used to confirm the biological activity of the NRGl modulators disclosed in the present invention.
A group of e.g. 9 women and 7 men, aged 30-66 years can be selected with documented MS progression during the 24 months before inclusion. Kurtzke's EDSS scores are between 3.0 (inclusive) and 7.5 (inclusive). All adverse events are documented; safety lab consists of serum transaminases (monthly for 3 months and every 3 months thereafter) and hematology (CBC and differential every 6 months) after the start of treatment. During the first year no specific treatment is given, during the second year a pharmaceutical composition according to this invention is given to all patients. MRI scanning consists of a 6-monthly inversion prepared 3D gradient echo sequence of the cervical cord, and yearly Tl- and T2 -weighted spin-echo sequences of the brain. The main efficacy parameter is the change in spinal cord cross-sectional area, obtained from 10 contiguous 3 -mm axial slices perpendicular to the cord above the center of the C2-C3. Scans are analysed in a randomized and blinded fashion. Stabilization or increase in cord area, reduction in Tl and T2 lesion load or change in EDSS score may identify positive results.

Claims

L A method of detecting the presence of or predisposition to multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in a subject, the method comprising detecting the presence of a susceptibility alteration in a NRGl gene or polypeptide in a sample from the subject, the presence of such an alteration being indicative of the presence of or predisposition to multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in said subject.
2. A method of assessing the response of a subject to a treatment of multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder, the method comprising detecting the presence of a susceptibility alteration in a NRGl gene or polypeptide in a sample from the subject, the presence of such an alteration being indicative of a responder subject.
3. The method according to claim 1 or 2, wherein said susceptibility alteration is a single nucleotide polymorphism (SNP).
4. The method according to any one of claims 1 to 3, wherein said susceptibility alteration is located within the 3' or 5' region of the NRGl gene.
5. The method of any one of claims 1 to 4, wherein the susceptibility alteration is selected from SNPs as listed in Table 2a, Table 2b or Table 2c.
6. The method according to any one of claims 1 to 5, wherein the presence of an alteration in the NRGl gene is detected by sequencing, selective hybridisation and/or selective amplification.
7. The use of a NRGl polypeptide or a nucleic acid encoding the same, for the manufacture of a pharmaceutical composition for treating or preventing multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in a subject. 8. The use according to claim 7 wherein the NRGl polypeptide has the sequence identified by SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 3 or SEQ ID NO. 4 or SEQ ID NO. 5 or SEQ ID NO. 6 or SEQ ID NO. 7 or SEQ ID NO.
8 or SEQ ID NO. 9 or SEQ ID NO. 10.
9. The use according to claim 8, wherein the NRGl polypeptide is a fusion protein between the Fc region of an antibody and a NRGl polypeptide defined by SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 3 or SEQ ID NO. 4 or SEQ ID NO. 5 or SEQ ID NO. 6 or SEQ ID NO. 7 or SEQ ID NO. 8 or SEQ ID NO. 9 or SEQ ID No. 10.
10. The use of any one of claim 7 to 9 for the manufacture of a pharmaceutical composition for treating or preventing multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in a subject presenting a susceptibility alteration in the NRGl gene or polypeptide.
11. The use of claim 10, wherein said susceptibility alteration is selected from the SNPs as listed in Table 2a, Table 2b, Table 2c, Table 2d or 2e.
12. The use of a NRGl gene or polypeptide as a target for screening candidate drugs for treating or preventing multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder.
13. A method of selecting biologically active compounds, said method comprising contacting a candidate compound with a NRGl gene or polypeptide and selecting compounds that bind said gene or polypeptide.
14. A method of selecting biologically active compounds, said method comprising contacting a candidate compound with recombinant host cell expressing a NRGl polypeptide, and selecting compounds that bind said NRGl polypeptide at the surface of said cells and/or that modulate the activity of said NRGl polypeptide.
15. The method of claim 13 or 14, further comprising a step of assaying the activity of the selected compounds in a model of multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder.
5 16. A method of treating multiple sclerosis or a related disorder in a subject in need of such treatment and wherein the subject has a susceptibility alteration in a NRGl gene comprising of administering to the subject a therapeutically effective amount of a medication for MS.
10 17. The method of claim 16 wherein the disorder related to MS is selected from progressive multifocal leukoencephalopathy (PML), acute disseminated encephalomyelitis (ADEM), acute demyelinating polyneuropathy (Guillain Barre syndrome), chronic inflammatory demyelinating neuropathy, Marchivafa-Bignami disease, central pontine myelinolysis, Devic syndrome, BaIo disease, HIV- or HTLV-myelopathy or a secondary
15 demyelinating disorder such as CNS lupus erythematodes, polyarteritis nodosa, Sjogren syndrome, sarcoidosis or isolated cerebral vasulitis.
18. The method of claim 16 or 17, wherein medication for MS is interferon-beta, preferably interferon-beta Ia.
20
19. The method of claim 16 to 18 wherein the susceptibility alteration is selected from one or more of the SNPs listed in Table 2a.
20. A method of treating systemic lupus erythematosus or a related disorder in a subject in 25 need of such treatment and wherein the subject has a susceptibility alteration in a NRGl gene comprising of administering to the subject a therapeutically effective amount of a medication for SLE.
21. The method of claim 20 wherein the related disorder is selected from cutaneous lupus, 30 drug-induced lupus, neonatal lupus and disorders such as rheumatoid arthritis, Sjogren's syndrome and scleroderma.
22. The method of claim 20 or 21 wherein the medication for SLE is selected from steroids such as corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), warfarin or antimalarial therapeutics such as quinacrine, hydroxychloroquine or chloroquine HCl.
5 23. The method of claim 20 to 22 wherein the susceptibility alteration is selected from one or more of the SNPs listed in Table 2b or Table 2d.
24. A method of treating psoriasis or a related disorder in a subject in need of such treatment and wherein the subject has a susceptibility alteration in a NRGl gene
10 comprising of administering to the subject a therapeutically effective amount of a medication for psoriasis.
25. The method of claim 24 wherein the related disorder is selected from psoriatic arthritis.
15 26. The method of claim 24 or 25 wherein the medication for psoriasis is selected from topical treatments, such as vitamin D3 analogs, corticosteroids, retinoids and coal tar, light therapy, methotrexate, oral retinoids, cyclosporin or biologies such as efalizumab.
27. The method of claim 24 to 26 wherein the susceptibility alteration is selected from one 20 or more of the SNPs listed in Table 2c or Table 2e.
28. A kit for the identification of a genetic polymorphism pattern at the NRGl gene associated with increased risk of the presence of or predisposition to multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in a subject, said kit
25 comprising:
(a) DNA sample collecting means, and
(b) means for determining a genetic polymorphism pattern for the NRGl gene.
29. An antibody, which selectively binds to NRGl polypeptide. 30
30. An antibody according to claim 29, which selectively binds to NRGl polypeptide having the sequence identified by SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 3 or SEQ ID NO. 4 or SEQ ID NO. 5 or SEQ ID NO. 6 or SEQ ID NO. 7 or SEQ ID NO. 8 or SEQ ID NO. 9 or SEQ ID NO.10.
31. A fusion protein between the Fc region of an antibody and a NRGl polypeptide defined by SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 3 or SEQ ID NO. 4 or SEQ ID NO. 5 or SEQ ID NO. 6 or SEQ ID NO. 7 or SEQ ID NO. 8 or SEQ ID NO. 9 or SEQ ID NO. 10.
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