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WO2006024169A1 - Genes de la region pseudoautosomale 1 et leur utilisation dans le diagnostic, le traitement et la prevention de maladies auto-immunes - Google Patents

Genes de la region pseudoautosomale 1 et leur utilisation dans le diagnostic, le traitement et la prevention de maladies auto-immunes Download PDF

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WO2006024169A1
WO2006024169A1 PCT/CA2005/001335 CA2005001335W WO2006024169A1 WO 2006024169 A1 WO2006024169 A1 WO 2006024169A1 CA 2005001335 W CA2005001335 W CA 2005001335W WO 2006024169 A1 WO2006024169 A1 WO 2006024169A1
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gene
seq
lymphocyte
group
lupus
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Pierre Chagnon
Lambert Busque
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HEMAX GENOME Inc
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HEMAX GENOME Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • 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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/104—Lupus erythematosus [SLE]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

Definitions

  • This invention relates to the modulation in the expression of genes located in the pseudoautosomal region 1 (PARl) and/or the activity of the proteins they encode, and particularly to the inhibition in the expression of genes located in PARl and/or the activity of the proteins they encode for treating, preventing and diagnosing autoimmune diseases.
  • PARl pseudoautosomal region 1
  • SLE Systemic Lupus Erythematosus
  • OMIM 152700 Systemic Lupus Erythematosus
  • Lymphopenia is also a typical feature of SLE and correlates with elevated disease activity and higher grades of systemic involvement (Wenzel et al., 2004; Crispin et al., 2003) .
  • SLE occurs in 40 to 50 per 100,000 people.
  • Familial aggregation (Kelly et al., 2002), monozygotic twin concordance (Grennan et al., 1997; Deapen et al., 1992), genetically engineered mutant mice which are susceptible to SLE (Waters, et al, 2001) , as well as the results of many genome scans support a genetic component of the disease.
  • a number of susceptibility loci for SLE have been found in different populations: namely, position 5pl5.3 (Namjou et al., 2002a), 10q22.3, 2q34-35, Ilpl5.6 (Quintero et al. , 2002), 19pl3.2, 18q21.1
  • SLE female predominance
  • the etiology for this predominance has been generally explained by the hormonal difference between males and females. The role of estrogens is probably a key in triggering SLE manifestations.
  • others have suggested an X-linked hypothesis to explain female predominance in SLE. Events such as X-inactivation which is specific to female could lead in a limited number of female to mosaicism between paternal and maternal X- linked genes in thymus which could lead to incomplete negative selection of T-cells leading to autoimmune manifestations.
  • the present invention relates to the treatment of autoimmune disease, more particularly to the modulation of expression or activity of genes located in a PARl region or the polypeptide they encode for the treatment or prevention of autoimmune diseases.
  • This modulation can also be used for the diagnosis or prognostication of autoimmune disease or for the screening of agents that can be used to treat or prevent autoimmune diseases.
  • a method of treating or preventing an autoimmune disease in a subject may comprise inhibiting the expression of a gene located in a PARl region and/or inhibiting the activity of the polypeptide encoded by the gene.
  • the gene is selected from the group consisting of CD99, IL3RA, SLC25A6, AK123701, FLJ13330, ASMTL, P2RY8, DXYS155E, ASMT, ZBEDl, BC019893 and AK096998.
  • the gene may comprise a nucleotide sequence substantially identical to the nucleotide sequence of SEQ ID NO: 3 or encode a polypeptide having the amino acid sequence of SEQ ID NO: 4.
  • the gene is IL3RA, it may comprise a nucleotide sequence substantially identical to the nucleotide sequence of SEQ ID NO: 5 or encode a polypeptide having the amino acid sequence of SEQ ID NO: 6.
  • the autoimmune disease is selected from the group consisting of Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison' s disease, ⁇ premature menopause, male infertility, myasthenia gravis, insulin- dependent diabetes mellitus, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, phacogenic uveitis, multiple sclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leukopenia, primary biliary cirrhosis, active chronic hepatitis, ulcerative colitis, Sjogren's syndrome, rheumatoid arthritis, scleroderma, Wegener's granulomatosis, dermatomyositis, lupus, atopic allergy, Lambert-Eaton syndrome
  • the disease when it is lupus, it may be selected, for example, from the group consisting of systemic lupus erythematosus, discoid lupus erythematosus, subacute cutaneous lupus erythematosus, drug-induced lupus and neonatal lupus.
  • the above-mentioned method comprises administering an agent capable of inhibiting the expression of the gene and/or the activity of the protein.
  • the gene may be located in a PARl region and/or the polypeptide may be encoded by the gene in an immune cell.
  • the immune cell can be selected from the group consisting of a lymphocyte, a dendritic cell, a monocyte, a macrophage, a neutrophil, an eosinophil, a basophil and a mast cell.
  • the lymphocyte may be selected from the group consisting of a T lymphocyte and a B lymphocyte.
  • the method can be applied to a mammal, and further to a human.
  • an agent capable of inhibiting the expression of a gene located in a PARl region and/or the activity of a polypeptide encoded by the gene for the treatment or prevention of an autoimmune disease in a subject.
  • the gene may be selected from the group consisting of CD99, IL3RA, SLC25A6, AK123701, FLJ13330, ASMTL, P2RY8, DXYS155E, ASMT, ZBEDl, BC019893 and AK096998.
  • the autoimmune disease is selected from the group consisting of Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison' s disease, premature menopause, male infertility, myasthenia gravis, insulin-dependent diabetes mellitus, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, phacogenic uveitis, multiple sclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leukopenia, primary biliary cirrhosis, active chronic hepatitis, ulcerative colitis, Sjogren's syndrome, rheumatoid arthritis, scleroderma, Wegener's granulomatosis, dermatomyositis, lupus, atopic allergy, Lambert-Eaton syndrome and mixed
  • the disease when it is lupus, it may be selected, for example, from the group consisting of systemic lupus erythematosus, discoid lupus erythematosus, subacute cutaneous lupus erythematosus, drug-induced lupus and neonatal lupus.
  • the agent may be adapted for administration to an immune cell.
  • the immune cell may be selected from the group consisting of a lymphocyte, a dendritic cell, a monocyte, a macrophage, a neutrophil, an eosinophil, a basophil and a mast cell.
  • the lymphocyte When the immune cell is a lymphocyte, the lymphocyte may be selected from the group consisting of a T lymphocyte and a B lymphocyte.
  • the use can performed in a mammal, and further, in a human.
  • a commercial package comprising an agent capable of inhibiting the expression of a gene located in a PARl region and/or the activity of a polypeptide encoded by the gene; and instructions for use of the agent in the treatment or prevention an autoimmune disease.
  • the gene may be selected from the group consisting of CD99, IL3RA, SLC25A6, AK123701, FLJ13330, ASMTL, P2RY8, DXYS155E, ASMT, ZBEDl, BC019893 and AK096998.
  • the autoimmune disease can be selected from the group consisting of Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison' s disease, premature menopause, male infertility, myasthenia gravis, insulin-dependent diabetes mellitus, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, phacogenic uveitis, multiple sclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leukopenia, primary biliary cirrhosis, active chronic hepatitis, ulcerative colitis, Sjogren's syndrome, rheumatoid arthritis, scleroderma, Wegener's granulomatosis, dermatomyositis, lupus, atopic allergy, Lambert-Eaton syndrome and
  • the disease when it is lupus, it may be selected, for example, from the group consisting of systemic lupus erythematosus, discoid lupus erythematosus, subacute cutaneous lupus erythematosus, drug-induced lupus and neonatal lupus.
  • the agent may be adapted for administration to an immune cell.
  • the immune cell can be selected from the group consisting of a lymphocyte, a dendritic cell, a monocyte, a macrophage, a neutrophil, an eosinophil, a basophil and a mast cell.
  • the immune cell when the immune cell is a lymphocyte, the lymphocyte may be selected from the group consisting of a T lymphocyte and a B lymphocyte.
  • an isolated nucleic acid comprising the sequence of SEQ ID NO: 29.
  • a pharmaceutical composition for use in the treatment or the prevention of an autoimmune disease.
  • the composition comprises an agent capable of inhibiting the expression of a gene located in a PARl region and/or the activity of a protein encoded by the gene; and a pharmaceutically acceptable carrier.
  • the gene may be selected from the group consisting of CD99, IL3RA, SLC25A6, AK123701, FLJ13330, ASMTL, P2RY8, DXYS155E, ASMT, ZBEDl, BC019893 and AK096998.
  • the composition can be adapted for administration to an immune cell.
  • the immune cell may be selected from the group consisting of a lymphocyte, a dendritic cell, a monocyte, a macrophage, a neutrophil, an eosinophil, a basophil and a mast cell.
  • the lymphocyte may be selected from the group consisting of a T lymphocyte and a B lymphocyte.
  • the method comprises assessing whether a test parameter is increased with respect to a control parameter.
  • the test parameter being selected from the group consisting of a copy number of a gene located in a PARl region, a level of expression of a gene located in a PARl region, a level of expression a protein encoded by a gene located in a PARl region, and an activity of a protein encoded by a gene located in a PARl region.
  • an increase in the test parameter is an indication that the subject has an increased likelihood of developing the autoimmune disease.
  • the gene is selected from the group consisting of CD99, IL3RA, SLC25A6, AK123701, FLJ13330, ASMTL, P2RY8, DXYS155E, ASMT, ZBEDl, BC019893 and AK096998.
  • the test parameter is measured from a biological sample obtained from said subject.
  • the biological sample can be a blood sample, and further a myeloid lymphocyte layer.
  • the autoimmune disease may be selected from the group consisting of Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison' s disease, premature menopause, male infertility, myasthenia gravis, insulin-dependent diabetes mellitus, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, phacogenic uveitis, multiple sclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leukopenia, primary biliary cirrhosis, active chronic hepatitis, ulcerative colitis, Sjogren's syndrome, rheumatoid arthritis, scleroderma, Wegener's granulomatosis, dermatomyositis, lupus, atopic allergy, Lambert-Eaton syndrome and
  • the disease when it is lupus, it may be selected, for example, from the group consisting of systemic lupus erythematosus, discoid lupus erythematosus, subacute cutaneous lupus erythematosus, drug-induced lupus and neonatal lupus.
  • control parameter may be selected from the group consisting of two copies of a gene located in a PARl region, a level of expression of a gene located in a PARl region in a control subject, a level of expression of a protein encoded by a gene located in a PARl region in a control subject and an activity of a protein encoded by a gene located in a PARl region in a control subject.
  • the control subject is a subject at an earlier time or a subject substantially free of said autoimmune disease.
  • the gene can be selected from the group consisting of CD99, IL3RA, SLC25A6, AK123701, FLJ13330, ASMTL, P2RY8, DXYS155E, ASMT, ZBEDl, BC019893 and AK096998.
  • the method may also comprise performing a polymerase chain reaction.
  • the test parameter and the control parameter is the copy number of said gene.
  • the polymerase chain reaction is a real-time quantitative polymerase chain reaction.
  • the test parameter and the control parameter is the level of expression of said gene.
  • the polymerase chain reaction is a reverse- transcriptase polymerase chain reaction.
  • a commercial package comprising means for assessing the expression of a gene located in a PARl region and/or the activity of a protein encoded by the gene and instructions for use of said means in the diagnosis or prognostication of an autoimmune disease in a subject.
  • the means is a set of primers selected from the group consisting of:
  • the method comprises assessing the ability of said agent of inhibiting the expression of a gene located in a PARl region and/or the activity of a protein encoded by the gene.
  • the inhibition is indicative that the agent is capable of treating or preventing the autoimmune disease.
  • the gene may be selected from the group consisting of CD99, IL3RA, SLC25A6, AK123701, FLJ13330, ASMTL, P2RY8, DXYS155E, ASMT, ZBEDl, BC019893 and AK096998.
  • the autoimmune disease is selected from the group consisting of Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison' s disease, premature menopause, male infertility, myasthenia gravis, insulin-dependent diabetes mellitus, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, phacogenic uveitis, multiple sclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leukopenia, primary biliary cirrhosis, active chronic hepatitis, ulcerative colitis, Sjogren's syndrome, rheumatoid arthritis, scleroderma, Wegener's granulomatosis, dermatomyositis, lupus, atopic allergy, Lambert-Eaton syndrome
  • the disease when it is lupus, it may be selected, for example, from the group consisting of systemic lupus erythematosus, discoid lupus erythematosus, subacute cutaneous lupus erythematosus, drug-induced lupus and neonatal lupus.
  • the assessment is performed in an immune cell.
  • the immune cell may be selected from the group consisting of a lymphocyte, a dendritic cell, a monocyte, a macrophage, a neutrophil, an eosinophil, a basophil and a mast cell.
  • the lymphocyte When the immune cell is a lymphocyte, the lymphocyte may be selected from the group consisting of a T lymphocyte and a B lymphocyte.
  • the method also comprises performing a polymerase chain reaction.
  • the polymerase chain reaction may be a reverse-transcriptase polymerase chain reaction.
  • a commercial package comprising means for assessing the expression of a gene located in a PARl region and/or the activity of a protein encoded by the gene and instructions for use of said means in screening for an agent capable of treating or preventing an autoimmune disease.
  • the gene is selected from the group consisting of CD99, IL3RA, SLC25A6, AK123701, FLJ13330, ASMTL, P2RY8, DXYS155E, ASMT, ZBEDl, BC019893 and AK096998.
  • the autoimmune disease is selected from the group consisting of Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison' s disease, premature menopause, male infertility, myasthenia gravis, insulin-dependent diabetes mellitus, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, phacogenic uveitis, multiple sclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leukopenia, primary biliary cirrhosis, active chronic hepatitis, ulcerative colitis, Sjogren's syndrome, rheumatoid arthritis, scleroderma, Wegener's granulomatosis, dermatomyositis, lupus, atopic allergy, Lambert-Eaton syndrome and mixed
  • the disease when it is lupus, it may be selected, for example, from the group consisting of systemic lupus erythematosus, discoid lupus erythematosus, subacute cutaneous lupus erythematosus, drug-induced lupus and neonatal lupus.
  • the instructions specify the use of an immune cell.
  • the immune cell may be selected from the group consisting of a lymphocyte, a dendritic cell, a monocyte, a macrophage, a neutrophil, an eosinophil, a basophil and a mast cell.
  • the lymphocyte When the immune cell is a lymphocyte, the lymphocyte may be selected from the group consisting of a T lymphocyte and a B lymphocyte.
  • the instructions specify the use of a polymerase chain reaction.
  • the polymerase chain reaction may be a reverse-transcriptase polymerase chain reaction.
  • FIG. 1 Karyotyping of the chromosomes of a patient having systemic lupus erythematosus (SLE) .
  • the patient possesses 22 intact autosomal chromosomes except for one X chromosome who harbours a small portion of the Y chromosome (arrow) .
  • Figure 2. Fluorescent in situ hybridization (FISH) analysis of the patient's metaphase chromosome.
  • FISH Fluorescent in situ hybridization
  • the Y specific gene SRY (sex determining region Y, pointed to by arrows) who localise normally on YpIl.31 is present on one of the X chromosome that is identified by the commercial fluorescence CEPX probe (identified with an asterix) , specific to the centromere of this chromosome (XpIl.1 to qll.l) .
  • FIG. 3 Y chromosome walking using specific PCR markers. Amplification was performed with the patient's blood DNA (lane 1), the patient's EBV transformed cells DNA (lane 2), normal men DNA (lanes 3 and 4) and normal women DNA (lane 5 and 6) . Markers A to D were detected in the patient's DNA. However, markers E to H were not detected in the patient's DNA suggesting that the translocation may have occurred between markers D and E. All markers were amplified in men DNA but not in women DNA showing the specificity of the markers used to walk the Y chromosome. The positions indicated under the figure correspond to the July 2003 assembly of the Human Genome Browser (UCSC) .
  • UCSC Human Genome Browser
  • FIG. 4 Alignment of the SLE male patient's breakpoint sequence (SEQ ID NO: 1) with the human chromosome YpIl.2 sequence (SEQ ID NO: 2) .
  • the XX male patient has 100% Y specific sequences for the first 473 nucleotide. After that position, an important number of variations are observed, indicating that the sequence may have switched to the X chromosome.
  • RepeatMasker http://www.repeatmasker.org
  • the rest of the X chromosome breakpoint sequence junction expending from 1001 to 1668 is shown in the lower part of the figure.
  • X chromosome sequence was obtained using inverse PCR.
  • Figure 5 Ideogram of the X chromosome (A), the Y chromosome (B) and the derivative X chromosome present in the XX male Lupus patient (C and D) harbouring 2 copies of the pseudoautosomal region 1 (PARl) in grey.
  • the genes located from IL3RA (1.100 mb) to CD99 (2.204 mb) in the PARl region are in two copies on the same chromosome. Arrows indicate the position of the breakpoint on the Xp22.33 and YpIl.2 chromosomes.
  • Figure 6. Graph showing the copy number of different genes of chromosome X for the Lupus patient and an average of four men and four women. Lined boxes represent the position of the three gaps remaining in the PARl region not yet sequence by the Human Genome Project.
  • Figure 7 Normalized expression levels of the CD99 mRNA in lymphocytes isolated from the patient (Ll) or from normal individuals (Nl to N6) .
  • Figure 8 Normalized expression levels of the CD99 mRNA in polymorphonuclear (PMN) cells isolated from the patient (Ll) or from normal individuals (Nl to N6) .
  • Figure 10 Human IL3RA coding sequence (SEQ ID NO: 5, Accession Number NM_002183) and polypeptide sequence (SEQ ID NO: 6, Accession Number NP_002174) .
  • Figure 11 Mouse IL3RA coding sequence (SEQ ID NO: 7, Accession Number X64534 S35630) and polypeptide sequence (SEQ ID NO: 8, Accession Number CAA45833) .
  • Figure 12. Human SLC25A6 coding sequence (SEQ ID NO: 9, Accession Number NM_001636) and polypeptide sequence (SEQ ID NO: 10, Accession Number NP_001627) .
  • Figure 13 Human AK123701 coding sequence (SEQ ID NO: 11, Accession Number AK123701) and polypeptide sequence (SEQ ID NO: 12, Accession Number BAC85679) .
  • Figure 14 Human FLJ13330 (or CXYorf2) coding sequence (SEQ ID NO: 13, Accession Number NM_025091) and polypeptide sequence (SEQ ID NO: 14, Accession Number NP_079367) .
  • Figure 15 Human ASMTL coding sequence (SEQ ID NO: 15, Accession Number NM_004192) and polypeptide sequence (SEQ ID NO: 16, Accession Number NP_004183) .
  • Figure 16 Human P2RY8 coding sequence (SEQ ID NO: 17, Accession Number NM_178129) and polypeptide sequence (SEQ ID NO: 18, Accession Number NP_835230) .
  • Figure 17. Human DXYS155E coding sequence (SEQ ID NO: 19, Accession Number NM_005088) and polypeptide sequence (SEQ ID NO: 20, Accession Number NP_005079) .
  • Figure 18 Human ASMT coding sequence (SEQ ID NO: 21, Accession Number NM_004043) and polypeptide sequence (SEQ ID NO: 22, Accession Number .
  • Figure 20 Human BC019893 coding sequence (SEQ ID NO: 25, Accession Number BC019893) and polypeptide sequence (SEQ ID NO: 26, Accession Number AAH19893) .
  • Figure 21 Human AK096998 coding sequence (SEQ ID NO: 27, Accession
  • Figure 22 Novel sequence from the X chromosome breakpoint, nucleotides 967 to 1668 from Figure 4 (SEQ ID NO: 29) .
  • the invention described herein relates to genes and proteins of the PARl region, especially to their use in the prevention, treatment and diagnosis of autoimmune diseases.
  • the invention relates to a method of treating or preventing an autoimmune disease.
  • treating or “treatment” is the alleviation of the manifestations or symptoms of the disease. More particularly, the treatment of an autoimmune disease implicates a reduction of the symptoms associated with such disease, a decrease in autoimmune reactions and/or a complete abrogation of autoimmunity.
  • the above-described method comprises inhibiting the expression of a gene located in a PARl region and/or the expression of a protein encoded by such gene.
  • “Inhibition” or “inhibiting” as used herein is intended to mean a reduction, a decrease or a complete abrogation of a specific activity (e.g. expression of a gene located in a PARl region and/or the activity of a polypeptide encoded by such gene) .
  • expression of a gene” and “activity of a protein” encompasses all the steps between the initial transcription of the gene (e.g. production of a corresponding mRNA) to the generation of a functional polypeptide or protein.
  • steps include, but are not limited to, the transcription of the gene into a transcript (e.g. mRNA), the transport of the transcript into the cytoplasm, the translation of the transcript into a polypeptide or protein, the maturation of the polypeptide or protein (e.g. post-translational modifications such as cleavage, glycosylation, folding) , the transport of the polypeptide or protein (e.g. to the nucleus, membrane, mitochondria, ER, Golgi, cytoplasm, etc), the activity of the polypeptide or protein (e.g. signalling (e.g. phosphorylation or association with another polypeptides), cleavage of a substrate, etc.) and the degradation of the polypeptide or protein.
  • a transcript e.g. mRNA
  • the transport of the transcript into the cytoplasm e.g. cytoplasm
  • the translation of the transcript into a polypeptide or protein e.g. post-translational modifications such as cleavage, glycosylation
  • the methods described herein can act on anyone of the above-mentioned steps directly or indirectly to inhibit the expression/activity of a gene or the polypeptide encoded by such gene.
  • the methods can act directly on the gene, the transcript of the gene and the polypeptide it encodes to inhibit their expression or activity.
  • the methods can also act indirectly on other location in the genome (e.g. promoter, enhancer, silencer, etc.), on other transcripts or on other polypeptides (e.g. ribonucleotide, enzyme, etc.) to achieve the desired inhibition.
  • polypeptide and “protein” are used interchangeably and designate a polypeptidic chain encoded by a gene.
  • the pseudoautosomal regions are located on the sex chromosomes and are divided into two distinct regions (PARl and PAR2) . These regions share a high degree of identity between the X and Y chromosome.
  • the rest of the sex chromosomes comprises sex-determining genes, which are usually distinct between the X and Y chromosome.
  • PARl region comprises different genes, which include but are not limited to, CD99 (SEQ ID NO: 3, Accession Number NM_002414), IL3RA (SEQ ID NO: 5, Accession Number NM_002183) , SLC25A6 (SEQ ID NO: 9, Accession Number NM_001636), AK123701 (SEQ ID NO: 11, Accession Number AK123701), FLJ13330 (or CXYorf2) (SEQ ID NO: 13, Accession Number NMJD25091), ASMTL (SEQ ID NO: 15, Accession Number NM_004192) , P2RY8 (SEQ ID NO: 17, Accession Number NM_178129) , DXYS155E (SEQ ID NO: 19, Accession Number NM_005088), ASMT (SEQ ID NO: 21, Accession Number NM_004043) , ZBEDl (SEQ ID NO: 23, Accession Number NM_004729) , BC019893 (SEQ ID NO: 25, Accession Number BC01
  • the CD99 gene is present in various organism, such as the green African monkey (nucleotide accession number U82166.1 and protein accession number AAB93833) .
  • sequence of the above-mentioned genes may vary from one individual to another (e.g. allelic variation).
  • allelic variation e.g. allelic variation
  • the methods described herein are not limited to the exact sequence of these genes (set forth, for example, in the figures) but encompass the allelic variants of these genes.
  • table 1 sets forth some allelic variants of the CD99 gene.
  • Table 1 Characterized single nucleotide polymorphims (SNPs) of the human CD99 gene
  • the invention described herein also relates to the inhibition of genes/proteins substantially identical to the genes/proteins of the PARl region.
  • the invention also relates to the inhibition of genes encoding polypeptides having a sequence homologous to polypeptides encoded by genes of the PARl region.
  • the invention further relates to the inhibition of the activity of the proteins encoded by such genes.
  • Homology and “homologous” refers to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing each position in the aligned sequences. A degree of homology between nucleic acid or between amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at positions shared by the sequences. As the term is used herein, a nucleic acid sequence is "homologous" to another sequence if the two sequences are substantially identical and the functional activity of the sequences is conserved (as used herein, the term “homologous” does not infer evolutionary relatedness) .
  • sequence similarity in optimally aligned substantially identical sequences may be at least 60%, 70%, 75%, 80%, 85%, 90% or 95%.
  • a given percentage of homology between sequences denotes the degree of sequence identity in optimally aligned sequences.
  • An "unrelated" or “non-homologous" sequence shares less than 40% identity, though preferably less than about 25 % identity, with any of SEQ ID NOs described herein.
  • Substantially complementary nucleic acids are nucleic acids in which the complement of one molecule is substantially identical to the other molecule. Two nucleic acid or protein sequences are considered substantially identical if, when optimally aligned, they share at least about 70% sequence identity. In alternative embodiments, sequence identity may for example be at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. MoI. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc.
  • Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al. ,
  • the BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold.
  • HSPs high scoring sequence pairs
  • Initial neighbourhood word hits act as seeds for initiating searches to find longer HSPs. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased.
  • Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • the BLAST program may use as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci. USA
  • nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • hybridisation to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in 0.2 x SSC/0.1% SDS at 42°C (see Ausubel, et al. (eds) , 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3) .
  • hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHPO 4 , 7% SDS, 1 mM EDTA at 65°C, and washing in 0.1 x SSC/0.1% SDS at 68°C (see Ausubel, et al. (eds), 1989, supra) .
  • Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology -- Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York) .
  • stringent conditions are selected to be about 5°C lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.
  • An immunological reaction can be defined as an event that implicates cells of the immune system (or immune cells) .
  • Cells of the immune system or “immune cells” are used herein interchangeably and designate cells concerned with immunity that are produced by the immune system.
  • autoimmunity relates to immunological events directed against the organism that produces it, and in addition, such events cause damage to the organism. Autoimmune reactions can be limited to a specific organ, tissue or cell type, can involve several organs, tissues or cell types or can be systemic.
  • Autoimmune diseases include, but are not limited to, Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison' s disease, premature menopause, male infertility, myasthenia gravis, insulin-dependent diabetes mellitus, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, phacogenic uveitis, multiple sclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leukopenia, primary biliary cirrhosis, active chronic hepatitis, ulcerative colitis, Sjogren's syndrome, rheumatoid arthritis, scleroderma, Wegener's granulomatosis, dermatomyositis, lupus, atopic allergy, Lambert-Eaton syndrom and mixed
  • lupus includes, but is not limited to, systemic lupus erythematosus, discoid lupus erythematosus, subacute cutaneous lupus erythematosus, drug-induced lupus and neonatal lupus.
  • the invention also relates to the administration of agents to treat or prevent autoimmune diseases.
  • the agent is capable of inhibiting expression/activity or a gene located in a PARl region or the protein encoded by such gene.
  • the agents selected can take many forms, such as a small molecule or drug, anti-sense nucleic acid molecule, a siRNA or RNAi, or a gene therapy that shuts down the expression of genes in the PARl region.
  • the agents can be used alone or in combination with other known agents used in conventional autoimmune therapies, such as nonsteroidal anti-inflammatory drugs (NSAIDS) (e.g. ibuprofen and naproxen), antimalarials (e.g. hydroxychloroquine); corticosteroids (e.g.
  • NSAIDS nonsteroidal anti-inflammatory drugs
  • ibuprofen and naproxen antimalarials
  • corticosteroids e.g.
  • prednisone hydrocortisone, methylprednisone or dexamethasone
  • immunosuppresives e.g. acyclophosphamide, mycophenolate mofetil, cyclosporine
  • antirheumatic drugs e.g. methotrexate
  • monoclonal antibodies directed against various cytokine such as monoclonal antibodies directed against tumor necrosis factor alpha, interleukin-1, or interleukin-2
  • gold salts e.g. methotrexate
  • the invention provides, for example, antisense molecules, interfering RNA or RNA-like molecules and ribozymes for exogenous administration to effect the degradation and/or inhibition of the translation of transcripts of genes of the PARl region.
  • therapeutic antisense oligonucleotide applications include: U.S. Pat. No. 5,135,917, issued Aug. 4, 1992; U.S. Pat. No. 5,098,890, issued Mar. 24, 1992; U.S. Pat. No. 5,087,617, issued Feb. 11, 1992; U.S. Pat. No. 5,166,195 issued Nov. 24, 1992; U.S. Pat. No. 5,004,810, issued Apr. 2, 1991; U.S.
  • the target mRNA for antisense binding may include not only the information to encode a protein, but also associated ribonucleotides, which for example form the 5'-untranslated region, the 3'-untranslated region, the 5' cap region and intron/exon junction ribonucleotides.
  • Antisense molecules (oligonucleotides) of the invention may include those which contain intersugar backbone linkages such as phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages, phosphorothioates and those with CH 2 --NH—O—CH 2 , CH 2 —N(CH 3 ) —0--CH 2 (known as methylene (methylimino) or MMI backbone), CH 2 —O—N(CH 3 ) —CH 2 , CH 2 —N(CH 3 ) —N(CH 3 ) —CH 2 and O—N(CH 3 ) —CH 2 —CH 2 backbones (where phosphodiester is O—P—0—CH 2 ) .
  • intersugar backbone linkages such as phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or
  • Oligonucleotides having morpholino backbone structures may also be used (U.S. Pat. No. 5,034,506) .
  • antisense oligonucleotides may have a peptide nucleic acid (PNA, sometimes referred to as "protein nucleic acid”) backbone, in which the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone wherein nucleosidic bases are bound directly or indirectly to aza nitrogen atoms or methylene groups in the polyamide backbone (Nielsen et al., 1991, Science 254:1497 and U.S. Pat. No. 5,539,082) .
  • the phosphodiester bonds may be substituted with structures which are chiral and enantiomerically specific. Persons of ordinary skill in the art will be able to select other linkages for use in practice of the invention.
  • Oligonucleotides may also include species which include at least one modified nucleotide base.
  • purines and pyrimidines other than those normally found in nature may be used.
  • modifications on the pentofuranosyl portion of the nucleotide subunits may also be effected. Examples of such modifications are 2'-O-alkyl- and 2'-halogen-substituted nucleotides.
  • n is from 1 to about 10; C 1 to Ci 0 lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF 3 ; OCF 3 ; O-, S-, or N-alkyl; 0-, S-, or N-alkenyl; SOCH 3 ; SO 2 CH 3 ; ONO 2 ; NO 2 ; N 3 ; NH 2 ; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the
  • the antisense oligonucleotides in accordance with this invention may comprise from about 5 to about 100 nucleotide units.
  • a nucleotide unit is a base- sugar combination (or a combination of analogous structures) suitably bound to an adjacent nucleotide unit through phosphodiester or other bonds forming a backbone structure.
  • RNAi RNA interference
  • expression of a nucleic acid encoding a polypeptide of interest, or a fragment thereof may be inhibited or prevented using RNA interference (RNAi) technology, a type of post-transcriptional gene silencing.
  • RNAi may be used to create a pseudo "knockout", i.e. a system in which the expression of the product encoded by a gene or coding region of interest is reduced, resulting in an overall reduction of the activity of the encoded product in a system.
  • RNAi may be performed to target a nucleic acid of interest or fragment or variant thereof, to in turn reduce its expression and the level of activity of the product which it encodes.
  • RNAi is described in for example Hammond et al. (2001), Sharp (2001), Caplen et al. (2001), Sedlak (2000) and published US patent applications 20020173478 (Gewirtz; published November 21, 2002) and 20020132788 (Lewis et al. ; published November 7, 2002) .
  • Reagents and kits for performing RNAi are available commercially from for example Ambion Inc. (Austin, TX, USA) and New England Biolabs Inc. (Beverly, MA, USA) .
  • RNAi The initial agent for RNAi in some systems is thought to be dsRNA molecule corresponding to a target nucleic acid.
  • the dsRNA is then thought to be cleaved into short interfering RNAs (siRNAs) which are 21-23 nucleotides in length (19-21 bp duplexes, each with 2 nucleotide 3' overhangs) .
  • the enzyme thought to effect this first cleavage step has been referred to as "Dicer” and is categorized as a member of the RNase III family of dsRNA-specific ribonucleases.
  • RNAi may be effected via directly introducing into the cell, or generating within the cell by introducing into the cell a suitable precursor (e.g.
  • RNA-induced silencing complex RISC
  • the RISC thus formed may subsequently target a transcript of interest via base-pairing interactions between its siRNA component and the target transcript by virtue of homology, resulting in the cleavage of the target transcript approximately 12 nucleotides from the 3' end of the siRNA.
  • RISC RNA-induced silencing complex
  • RNAi may be effected by the introduction of suitable in vitro synthesized siRNA or siRNA-like molecules into cells. RNAi may for example be performed using chemically-synthesized RNA (Brown et al., 2002) . Alternatively, suitable expression vectors may be used to transcribe such RNA either in vitro or in vivo. In vitro transcription of sense and antisense strands (encoded by sequences present on the same vector or on separate vectors) may be effected using for example T7 RNA polymerase, in which case the vector may comprise a suitable coding sequence operably-linked to a T7 promoter. The in vitro-transcribed RNA may in embodiments be processed (e.g. using E.
  • RNA duplex which is introduced into a target cell of interest.
  • Other vectors may be used, which express small hairpin RNAs (shRNAs) which can be processed into siRNA-like molecules.
  • shRNAs small hairpin RNAs
  • siRNA-like molecule refers to a nucleic acid molecule similar to an siRNA (e.g. in size and structure) and capable of eliciting siRNA activity, i.e. to effect the RNAi-mediated inhibition of expression.
  • such a method may entail the direct administration of the siRNA or siRNA-like molecule into a cell, or use ' of the vector-based methods described above.
  • the siRNA or siRNA-like molecule is less than about 30 nucleotides in length.
  • the siRNA or siRNA-like molecule is about 21-23 nucleotides in length.
  • siRNA or siRNA- like molecule comprises a 19-21 bp duplex portion, each strand having a 2 nucleotide 3' overhang.
  • the siRNA or siRNA-like molecule is substantially identical to a nucleic acid encoding a polypeptide of interest, or a fragment or variant (or a fragment of a variant) thereof. Such a variant is capable of encoding a protein having activity similar to the polypeptide of interest.
  • the sense strand of the siRNA or siRNA-like molecule is substantially identical to SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21 or a fragment thereof (RNA having U in place of T residues of the DNA sequence) .
  • the agents described herein can also be adapted for administration to an immune cell. The agents can be modified for targeting to an immune cell and/or for acting preferentially on an immune cell.
  • lymphocyte e.g. B and T lymphocytes
  • dendritic cell monocyte
  • macrophage e.g. monocyte
  • neutrophil e.g. polymorphonuclear cell or PMN
  • eosinophil basophil and mast cell.
  • the subject is a mammal, and further, a human.
  • the invention also relates to the use of the agent described above to treat or prevent autoimmune diseases in a subject (e.g. mammal or human) .
  • the agent is capable of inhibiting expression/activity of a gene located in a PARl region and/or the activity of the protein encoded by such gene.
  • the gene located in the PARl region can be selected from the group consisting of CD99, IL3RA, SLC25A6, AK123701, FLJ13330, ASMTL, P2RY8, DXYS155E, ASMT, ZBEDl, BC019893 and AK096998.
  • the agent may also be adapted from administration to an immune cell. Corresponding commercial packages reflecting the uses set forth herein are also contemplated.
  • the invention relates to an isolated nucleic acid comprising the following sequence (nucleotides 967 to 1668 from Figure 4) :
  • Such sequence or fragments thereof can be used, for example, to screen for the presence of a breakpoint on the X chromosome (e.g. as a probe, for the design of oligonucleotides, primers, etc., refer to the diagnostic methods mentioned below) .
  • this novel sequence since this novel sequence is located in one of the gaps on the human X chromosome (refer to the Examples below) , this novel DNA sequence may also serve to further characterize (e.g. sequence) such gap (e.g. probe, primers, oligonucleotides, etc.) .
  • an isolated nucleic acid, or homolog, fragment or variant thereof may further be incorporated into a recombinant expression vector.
  • the vector will comprise transcriptional regulatory sequences or a promoter operably-linked to a nucleic acid comprising a sequence capable of encoding a peptide compound, polypeptide or domain of the invention.
  • a first nucleic acid sequence is "operably-linked" with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably-linked to a coding sequence if the promoter affects the transcription or expression of the coding sequences.
  • operably-linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame.
  • Transcriptional regulatory element is a generic term that refers to DNA sequences, such as initiation and termination signals, enhancers, and promoters, splicing signals, polyadenylation signals which induce or control transcription of protein coding sequences with which they are operably-linked.
  • the recombinant expression vector of the present invention can be constructed by standard techniques known to one of ordinary skill in the art and found, for example, in Sambrook et al. (1989) in
  • the vectors of the present invention may also contain other sequence elements to facilitate vector propagation and selection in bacteria and host cells.
  • the vectors of the present invention may comprise a sequence of nucleotides for one or more restriction endonuclease sites. Coding sequences such as for selectable markers and reporter genes are well known to persons skilled in the art.
  • a recombinant expression vector comprising a nucleic acid sequence of the present invention may be introduced into a host cell, which may include a living cell capable of expressing the protein coding region from the defined recombinant expression vector.
  • the living cell may include both a cultured cell and a cell within a living organism.
  • the invention also provides host cells containing the recombinant expression vectors of the invention.
  • host cell and "recombinant host cell” are used interchangeably herein. Such terms refer not. only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used ⁇ herein.
  • Vector DNA can be introduced into cells via conventional transformation or transfection techniques.
  • transformation and “transfection” refer to techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection and viral-mediated transfection. Suitable methods for transforming or transfecting host cells can for example be found in Sambrook et al. (supra), and other laboratory manuals. Methods for introducing DNA into mammalian cells in vivo are also known, and may be used to deliver the vector DNA of the invention to a subject for gene therapy for an autoimmune disease.
  • the invention also relates to a pharmaceutical composition to be used in the treatment or prevention of an autoimmune disease.
  • the composition comprises an agent capable of inhibiting expression/activity or a gene located in a PARl region and/or the polypeptide such gene encodes as well as a pharmaceutically acceptable carrier.
  • the agent can be adapated for administration to an immune cell.
  • the invention provides agents that are purified, isolated or substantially pure.
  • An agent is "substantially pure” when it is separated from the components that naturally accompany it.
  • an agent is substantially pure when it is at least 60%, more generally 75% or over 90%, by weight, of the total material in a sample.
  • a polypeptide that is chemically synthesised or produced by recombinant technology will generally be substantially free from its naturally associated components.
  • a nucleic acid molecule is substantially pure when it is not immediately contiguous with (i.e., covalently linked to) the coding sequences with which it is normally contiguous in the naturally occurring genome of the organism from which the DNA of the invention is derived.
  • a substantially pure agent can be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid molecule encoding a polypeptide compound; or by chemical synthesis. Purity can be measured using any appropriate method such as column chromatography, gel electrophoresis,
  • agents described herein may be used therapeutically in formulations or medicaments to prevent or treat autoimmune disease.
  • the invention provides corresponding methods of medical treatment, in which a therapeutic dose of the agent is administered in a pharmacologically acceptable formulation, e.g. to a patient or subject in need thereof.
  • a therapeutic dose of the agent is administered in a pharmacologically acceptable formulation, e.g. to a patient or subject in need thereof.
  • such compositions include the agent in a therapeutically or prophylactically effective amount sufficient to treat an autoimmune disease.
  • the therapeutic composition may be soluble in an aqueous solution at a physiologically acceptable pH.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as a reduction of expression/activity of a gene located in a PARl region and/or the polypeptide it encodes and in turn a reduction in autoimmune disease progression.
  • a therapeutically effective amount of an agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the agent to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the agent are outweighed by the therapeutically beneficial effects.
  • a prophylactically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as preventing or inhibiting the rate of autoimmune disease onset or progression.
  • a prophylactically effective amount can be determined as described above for the therapeutically effective amount.
  • specific dosage regimens may be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for parenteral administration.
  • the carrier can be suitable for intravenous, intraperitoneal, intramuscular, sublingual or oral administration.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the agent, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.
  • an agent can be administered in a time release formulation, for example in a composition which includes a slow release polymer.
  • the agents can be prepared with carriers that will protect the agent against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG) . Many methods for the preparation of such formulations are patented or generally known to those skilled in the art.
  • Sterile injectable solutions can be prepared by incorporating the agent in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the agent into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • an agent may be formulated with one or more additional compounds that enhance the solubility of the agent.
  • the methods or assays for the assessment of autoimmune diseases can indicate broadly the presence of autoimmunity or can indicate the presence of a specific disease.
  • the presence of auto-antibodies in the subject's fluid, cells, tissues or organs is usually indicative of an underlying autoimmune disease.
  • "Auto-antibodies" are antibodies that target one's own cell, tissue or organ. Whenever specific auto-antibodies are linked to a specific disease whereas others are linked to a general auto-immune response.
  • auto-antibodies can be detected in a biological sample obtained from the subject, such as a fluid (e.g. urine, tears, blood, serum or myeloid lymphocyte layer) or a biopsy (such as a kidney biopsy or a skin biopsy) .
  • a biological sample obtained from the subject
  • Such auto-antibodies include, but are not limited to, anti-dsDNA antibodies, anti-nuclear antiboby (ANA or anti-DNA) , anti-Smith (anti-Sm) , anti-ribonucleoprotein (anti-RNP), anti-Ro (SSA), anti-La (SSB) and anticardiolipin (or antiphospholipid) antibody.
  • the auto-antibodies can be visualized alone or in combination with the antigen it recognises.
  • the auto-antibodies can be visualized by many techniques known by those ordinary skilled in the art such as immunofluorescence staining, ELISA, RIA, X rays and other imaging techniques.
  • routine laboratory tests can also be performed to assess the presence or severity of an autoimmune disease.
  • blood count, urinalysis, blood chemistries, erythrocyte sedimentation rate and complement can be used to diagnose SLE or to measure the severity of the disease.
  • the presence of certains symptoms can also help a physician to diagnose an autoimmune disease.
  • people suffering from SLE can experience extreme fatigue, painful or swollen joints (e.g. arthritis), unexplained fever, skin rashes, chest pain (e.g. pleuritis) , unusual hair loss, pale or purple fingers or toes (e.g. Raynaud's phenomenon), sun sensitivity, edema in legs and around the eyes, mouth ulcers, nephritis, central nervous system disorders (e.g. headaches dizziness, memory disturbances, vision problems, seizures, stroke or change in behavior) , inflammation of the blood vessels (e.g.
  • vasculitis anemia, leukopenia, thrombocytopenia, inflammation of the heart (e.g. myocarditis or endocarditis) or the heart membrane (e.g. pericarditis) and/or atherosclerosis.
  • myocarditis or endocarditis e.g. myocarditis or endocarditis
  • pericarditis e.g. pericarditis
  • the present invention also relates to a method of diagnosing or prognosticating an autoimmune disease.
  • This method comprises assessing whether a test parameter is altered (e.g. increased) with respect to a control parameter. Such alteration (e.g. increase) is indicative of an increased likelihood of developing an autoimmune disease.
  • a test parameter can be selected from the group consisting of a gene copy number, a level of expression of a gene, a level of expression of a protein encoded by a gene and a level of protein activity.
  • the gene is a gene located in a PARl region and the protein is a protein encoded by such gene.
  • the gene is selected from the group consisting of CD99, IL3RA, SLC25A6, AK123701, FLJ13330, ASMTL, P2RY8, DXYS155E, ASMT, ZBEDl, BC019893 and AK096998.
  • the gene copy number can be assessed by using PCR (e.g. real-time quantitative PCR) or karyotyping.
  • the level of genetic expression can be measured with techniques such as PCR (e.g. RT-PCR, DD-PCR, subtractive PCR), Northern blotting or DNA micro-array.
  • the level of protein expression can be quantified by Western blotting, 2D electrophoresis, capillary electrophoresis, imaging techniques (e.g. specific antibodies coupled to immunofluorescent compounds or enzyme that enable colorimetric visualisation) , ELISA, RIA, protein micro-array.
  • the assessment of protein activity is specific to the protein that is studied.
  • the activity of the protein encoded by the CD99 gene can be measured by conventional techniques for the measurement of apoptosis known to one skilled in the art.
  • Such techniques include, but are not limited to, TUNEL staining, Annexin V staining, FACS analysis, agarose electrophoresis, Western blot, histology, electron microscopy, ELISA, mitochondrial assay (e.g.
  • IL3RA is associated with the proliferation and differentiation of stem cell into neutrophil, basophil, eosinophil, monocyte and bone marrow precursors.
  • IL3RA-induced proliferation can be measured, for example, using BrdU staining, FACS cell cycle analysis.
  • IL3RA-induced differentiation can be measured, for example, with antibodies directed against differentiation-specific markers.
  • specific antibodies directed against CD3 can be used to identify T lymphocytes, against CD14 to identify monocytes, against CD19 to identify B lymphocytes and against CD34 to identify hematopoietic stem cells.
  • SLC25A6 is implicated in the exhange of ADP and ATP across the mitochondrial membrane. As such, SLC25A6 activity can be assessed, for example, by comparing the mitochodrial content of ADP and ATP to the - cytoplasmic content of ADP and ATP.
  • ASMT is involved in the biosynthesis of melatonin. As such, ASMT activity can be measured, for example, by assessing the amount of melatonin produced.
  • ZBEDl is a zinc finger protein that binds specifically to promoter sequences and modulates the expression of genes linked to the promoter. As such,
  • ZBEDl activity can be assessed via a transcriptionnal assay, such as the beta-galactosidase assay or the luciferase assay.
  • the test parameter can be assessed in a biological sample obtained from a subject.
  • the biological sample can be, for example, blood, a blood fraction (such as serum or the myeloid lymphocyte layer) , urine, tears or any other biological fluid.
  • the biological sample can also be a biopsy of an organ (e.g. kidney or skin) or tissue that is suspected to be affected by an autoimmune reaction.
  • the value of the test parameter may be compared to the value of a control parameter.
  • Such control parameter include, but is not limited to, two copies of a gene located in a PARl region, a level of expression of a gene located in a PARl region in a control subject, a level of a protein encoded by a gene located in a
  • a control subject is considered as a subject at an earlier time and/or a subject not affected by an autoimmune disease (e.g. substantially free of one or more autoimmune disease) .
  • the assessment of the alteration (e.g. increase) in the test parameter can include, for example, a polymerase chain reaction (PCR) .
  • the polymerase chain reaction may be a reverse- transcriptase polymerase chain reaction or real-time polymerase chain reaction. The latter can be performed, for example, with one or more of the following set of primers:
  • the above-mentioned diagnostic method can also be adapted to detect a translocation between the X and the Y chromosome, such translocation causing a triplication (e.g. a partial triplication) of genes located in the PARl region.
  • a Southern blot may be performed on the subject's genomic DNA. Such Southern blot may be performed using a probe capable of hybridizing on both sides of the translocation site.
  • the probe can optionnally be the nucleotide sequence set forth in Figure 4 (e.g. SEQ ID NO: 1) or a fragment thereof. Under stringent conditions, the probe should hybridize to the DNA of a subject harbouring the translocation.
  • a PCR could also be performed with the subject's genomic DNA using primers located on either side of the translocation.
  • one of the' primers used to perform the PCR reaction is located on the X chromosome, the other being located on the Y chromosome. These primers will only generate a PCR amplification product in subjects harbouring the translocation. These primers may be, for example, the following: 5'-AATACTGTCTTCTCCCCAATG-S' (SEQ ID NO: 50) and 5'-CGTCCTTGTCACCGCCAACTG-S' (SEQ ID NO 51) .
  • multiple sets of primers can be used to perform the PCR reaction and determine the presence of a translocation.
  • the sets of primers are specific to one of the sex chromosome (e.g. the Y chromosome) and are located on either side of the translocation.
  • the first set of primers will generate a PCR amplification product in normal males and the subject harbouring the translocation, but not in normal females.
  • the second set of primers will generate a PCR amplification product only in normal males, but not in subject harbouring the translocation or females.
  • the first of primers that can be used may have the following sequences:
  • the second set of primers may have the following sequences: 5'-TGAACCACACTTGCATCACTG-S' (SEQ ID NO: 38) and 5'-AAAAGTCTTCCAGTCCATAAC-S' (SEQ ID NO: 39) .
  • a commercial package comprising means to assess expression/activity of a gene located in a PARl region or the protein it encodes and instructions for its use in the diagnosis or prognostication of an autoimmune disease in a subject.
  • Many different means can be designed by one skilled in the art to assess such expression and/or activity.
  • the above-mentioned primers can serves as detection means.
  • the invention also provides a method of screening for an agent capable of treating or preventing an autoimmune disease, such as those mentioned above.
  • the screening method may optionally comprise assessing the ability of the agent to inhibit expression/activity of a gene located in a PARl region or the polypeptide it encodes.
  • the screening can be performed, for example, in an immune cell.
  • immune cell may be selected from the group consisting of a lymphocyte (e.g. B or T lymphocyte) , a dendritic cell, a monocyte, a macrophage, a neutrophil (e.g. PMN), an eosinophil, a basophil and a mast cell.
  • the screening can be performed in a transgenic animal overexpression a gene located in a PARl region.
  • the invention also relates to a transgenic animal (e.g. rodent) that overexpresses a gene located in a PARl region.
  • This transgenic animal can be used as a model to study autoimmunity (e.g. SLE) .
  • the transgenic animal can also be used to screen for agents or compounds that can be used in the treatment or prevention of autoimmune diseases.
  • the agent screened with this method may be used to treat or prevent one or more autoimmune diseases (for examples of autoimmune diseases, refer to the list above) .
  • autoimmune diseases for examples of autoimmune diseases, refer to the list above.
  • the techniques used to assess the inhibition are known to one skilled in the art and may comprise one or more of the techniques described above.
  • the invention further provides a method of identifying an agent for the prevention and/or treatment of autoimmune diseases based on the identification of an agent capable of modulating (e.g. inhibiting) the expression/activity of a gene located in a PARl region or the polypeptide it encodes.
  • a method may comprise assaying gene expression in the presence versus the absence of a test agent.
  • gene expression may be measured by detection of the corresponding RNA or protein, or via the use of a suitable reporter construct comprising a transcriptional regulatory element (s) normally associated with such gene, operably-linked to a reporter gene.
  • a first nucleic acid sequence is "operably-linked" with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably- linked to a coding sequence if the promoter affects the transcription or expression of the coding sequences.
  • operably-linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame.
  • enhancers generally function when separated from the promoters by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably-linked but not contiguous.
  • Transcriptional regulatory element is a generic term that refers to DNA sequences, such as initiation and termination signals, enhancers, and promoters, splicing signals, polyadenylation signals which induce or control transcription of protein coding sequences with which they are operably-linked.
  • the expression of such a reporter gene may be measured on the transcriptional or translational level, e.g. by the amount of RNA or protein produced.
  • RNA may be detected by for example Northern analysis or by the reverse transcriptase-polymerase chain reaction (RT- PCR) method (see for example Sambrook et al (1989) Molecular Cloning: A Laboratory Manual (second edition) , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA) .
  • Protein levels may be detected either directly using affinity reagents (e.g. an antibody or fragment thereof [for methods, see for example Harlow, E. and Lane, D (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY] ; a ligand which binds the protein) or by other properties (e.g. fluorescence in the case of green fluorescent protein) or by measurement of the protein' s activity, which may entail enzymatic activity to produce a detectable product (e.g. with altered spectroscopic properties) or a detectable phenotype (e.g. alterations in cell growth) .
  • Suitable reporter genes include but are not limited to chloramphenicol acetyltransferase, beta-D galactosidase, luciferase, or green fluorescent protein.
  • the above-noted methods and assays may be employed either with a single test compound or a plurality or library (e.g. a combinatorial library) of test compounds. In the latter case, synergistic effects provided by combinations of compounds may also be identified and characterized.
  • the above-mentioned compounds may be used for inhibiting the expression/activity of a gene located in a PARl region or the polypeptide it encodes, and for the prevention and/or treatment of autoimmune diseases, or may be used as lead compounds for the development and testing of additional compounds having improved specificity, efficacy and/or pharmacological (e.g. pharmacokinetic) properties.
  • one or a plurality of the steps of the screening/testing methods of the invention may be automated.
  • Such assay systems may comprise a variety of means to enable and optimize useful assay conditions.
  • Such means may include but are not limited to: suitable buffer solutions, for example, for the control of pH and ionic strength and to provide any necessary components for optimal protein activity and stability (e.g. protease inhibitors), temperature control means for optimal protein activity and or stability, and detection means to enable the detection of the protein activity.
  • detection means may be used, including but not limited to one or a combination of the following: radiolabelling (e.g. 32 P), antibody-based detection, fluorescence, chemiluminescence, spectroscopic methods (e.g. generation of a product with altered spectroscopic properties), various reporter enzymes or proteins (e.g.
  • binding reagents e.g. biotin/ (strept)avidin
  • Binding may also be analysed using generally known methods in this area, such as electrophoresis on native polyacrylamide gels, as well as fusion protein-based assays such as the yeast 2-hybrid system or in vitro association assays, or proteomics- based approachs to identify binding proteins.
  • the assay may be carried out in vitro utilizing a host cell which may comprise naturally isolated or recombinantly produced proteins, in preparations ranging from crude to pure. Recombinant proteins may be produced in a number of prokaryotic or eukaryotic expression systems which are well known in the art. Such assays may be performed in an array format. In certain embodiments, one or a plurality of the assay steps are automated. A homolog, variant and/or fragment of a protein encoded by a gene located in a PARl region which retains activity may also be used in the methods of the invention.
  • Homologs includes protein sequences which are substantially identical to the amino acid sequence of a protein encoded by a gene located in a PARl region, sharing significant structural and functional homology with such protein.
  • Variants include, but are not limited to, proteins or peptides which differ by any modifications, and/or amino acid substitutions, deletions or additions.
  • Such variants include fusion proteins, for example a protein of interest or portion thereof fused with a suitable fusion domain (such as glutathione-S-transferase fusions, and others) .
  • Modifications can occur anywhere including the polypeptide backbone, (i.e. the amino acid sequence), the amino acid side chains and the amino or carboxy termini.
  • substitutions, deletions or additions may involve one or more amino acids.
  • Fragments include a fragment or a portion of a protein or a fragment or a portion of a homolog or variant of a protein.
  • the assay may in an embodiment be performed using an appropriate host cell.
  • a host cell may be prepared by the introduction of DNA into the host cell and providing conditions for the expression of genes located in a PARl region.
  • host cells may be prokaryotic or eukaryotic, bacterial, yeast, amphibian or mammalian.
  • the above-described assay methods may further comprise determining whether any compounds so identified can be used for the prevention or treatment of autoimmune diseases, such as examining their effect (s) on disease symptoms in suitable autoimmune disease animal model systems.
  • Also contemplated herein is a commercial package comprising means to assess expression/activity of a gene located in a PARl region or the protein it encodes and instructions for its use in the screening of agents capable of treating or preventing an autoimmune disease in a subject.
  • Many different means can be designed by one skilled in the art to assess such expression and/or activity.
  • the above- mentioned primers can serves as detection means.
  • Example 1 Material and methods
  • Lymphocytes from the patient were transformed and immortalized with Epstein Barr virus (EBV) . Those cells were used as a source a genomic DNA and also for metaphase chromosome used for Fluorescence In Situ Hybridisation (FISH) . The transformed cells were cultured in Iscove medium.
  • EBV Epstein Barr virus
  • FISH Fluorescence In situ Hybridisation
  • SKY human Spectral Karyotyping
  • FISH FISH. All probes were first used in FISH on chromosome preparations of normal metaphases to confirm their predicted localization (for details see, http://www.biologia.uniba.it/rhmc) . Both female and male normal metaphases were used. Digital images were obtained using a fluorescence microscope ZEIZZ AXIOSCOP 2 PLUS equipped with a cooled CCD camera. FITC (green), rhodamine (red-orange) and 4, 6- diamidino-2-phenyl-indole (DAPI, blue) fluorescence signals, detected using specific filters. Pseudocoloring and merging of images were performed by CytoVisionTM System software (Applied Imaging, Santa Clara, CA) .
  • Microsatellite markers analysis 54 highly polymorphic markers that span the entire X chromosome surface have been genotyped. All markers came from the ABI panel (Applied Biosystems) . PCR primers were labelled with 6-FAM, NED or HEX phosphoramidite. PCR were performed on 2700 ABI thermocyclers. Pool products were run on a 3100 sequencer (ABI), and the results were analyzed by Genescan (v.2.0) and Genotyper (v.2.1) software, to derive allele sizes. Y chromosome specific PCR.
  • BACs Y bacterial artifical chromosomes
  • Oligonucleotide sequences used in the Y chromosome of the marker A forward, 5'-ATTTTATGACTATACAAACCTGC-S' (SEQ ID NO: 30) and reverse, 5'-TCTCTAAATACACTTGTTAGTAA-S' (SEQ ID NO: 31), annealing temperature 50°C, chromosome Y position 4.15 Mb; B: forward, 5'-ATGCCTTCATCAGGATGCTA-S' (SEQ ID NO: 32) and reverse, 5'- GCTAGTATTTTGTTATGGACTTT-3' (SEQ ID NO: 33), annealing temperature 50 0 C, chromosome Y position 4.19 Mb; C: forward, 5'-ACAGTGTATCACATTTTAGTCC-S' (SEQ ID NO: 34) and reverse, 5'-ATCACTACTGTCTACTATGAGC-S' (SEQ ID NO: 35), annealing temperature 50 0 C, chromosome Y position 4.197 Mb;
  • Genomic DNA samples (2 ⁇ g) , from a normal individuals and the patient, were digested overnight (0/N) at 37°C with 30 U of Kpn ⁇ and 2 ⁇ l of the appropriate buffer (NEB) in a total volume of 20 ⁇ l. The digestion was first conducted with half of the enzyme quantity for 2 hours, before adding the remainder of enzyme and pursuing the digestion. The reactions were phenol-chloroform extracted, ethanol precipitated and resuspended in 30 ⁇ l of ddH 2 O.
  • Intramolecular ligation of the Kpnl fragments was performed by using the totality of the digestion products with 200 U of T4 DNA ligase and 50 ⁇ l of the appropriate buffer (NEB) in a total volume of 500 ⁇ l. Extra ATP was added to a final concentration of 1 mM. The reactions were incubated at 16 C C 0/N. The ligation products -were ethanol precipitated and resuspended in 15 ⁇ l of ddH 2 O. Two rounds of PCR were conducted using nested primers for the second round.
  • PCR reactions were done in a total volume of 50 ⁇ l, with 2.5 U of Taq polymerase, the appropriate buffer (Sigma) and final concentrations of 0.45 ⁇ M of each primer, 1.5 mM of MgCl 2 , 0.4 mM of each dNTP, 4 % of formamid and 10 % of glycerol.
  • the parameters for the amplifications were as followed: a first denaturation step of 5 min at 95°C, followed by 45 cycles of 45 sec at 95°C, 30 sec at 50 0 C and 3 min 30 sec at 72 0 C, and a final elongation step of 7 min at 72 0 C.
  • the region of the breakpoint was obtain by using, in the same PCR reaction, a forward primer specific to the Y chromosome 5'-AATACTGTCTTCTCCCCAATG-S' (SEQ ID NO: 50) and a reverse primer specific to the X chromosome 5'-CGTCCTTGTCACCGCCAACTG-S' (SEQ ID NO: 51) .
  • ABI Prism 7000 SDS was programmed to an initial step of 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C. All reactions were run in triplicate.
  • Real-time quantitative PCR The gene copy number of various markers located in the PARl region was determined for the patient and normal individuals by quantitative real-time PCR on a ABI Prism 7000 Sequence Detection System (Applied Biosystems, USA) . DNA was extracted from the myeloid lymphocyte layer from the patient and the control.
  • PCR reactions were carried out on 12.5-25 ng of DNA. For all standard curves, 4 DNA dilutions ranging from 2.5 to 100 ng (825 to 33 000 gene copies) were used. The same DNA dilution of any given sample was used across all reactions . All PCRs were performed using SYBRTM Green PCR Master Mix (Applied Biosystems, USA) in a total volume of 25 ⁇ l.
  • each primer was 200 nM.
  • ABI Prism 7000 SDS was programmed to an initial step of 10 min at 95°C, followed by 40 cycles of 15 sec at 95 1 C and 1 min at 60"C. All reactions were run in triplicate, in two to three independent assays. The mean gene copy numbers were calculated from these assays and used to calculate N values. Table 2 sets forth the primers used for this assay:
  • ANA positive antinuclear antibodies
  • Sm anti-DNA
  • Sm smooth muscle
  • TTP protracted thrombotic thrombocytopenic purpura
  • Treatment of the patient's disease has included corticosteroids, hydroxychloroquine, methotrexate, intravenous pulse cyclophosphamide, azathioprine and plasmapheresis.
  • the patient has required treatment for hypertension and has received pamidronate for osteoporosis.
  • the abnormal chromosome contains the long arm (q) and most of the short arm (p) of the X chromosome (p22.3 to qter) and a portion of the terminal short arm of a Y chromosome (pter to pll.2).
  • FISH analysis confirmed that the derivative X chromosome contained both the X chromosome centromere (CEPX) and the Y chromosome's SRY gene (Fig. 2) . This gene was thus transferred to the X chromosome and confirmed that the translocation had occurred downstream of this gene on the Y chromosome (2.30 mb according to the July 2003 assembly of the UCSC Genome Browser, http://genome.ucsc.edu/) .
  • microsatellite markers that span the entire X chromosome were analyzed. Heterozygosity at microsatellite markers indicates the presence of two X chromosomes, whereas homozygosity suggests the presence of only one chromosome (loss of heterozygosity) . An analysis of the patient's genomic DNA revealed an heterozygosity for most of these markers, indicating the presence of almost two entire X chromosomes. Starting from the short arm (p) telomere, the first heterozygote marker detected is GATA164D10 (position 2.96 mb) .
  • the X chromosome breakpoint has to be located between the telomere and this position, probably in the PARl region.
  • GATA164D10 is located upstream of PRKX gene, which eliminates the possibility that the translocation occurred by homologous recombination between PRKX and PRKY genes, the usual X:Y translocation breakpoint in XX male (Wang et al., 1995) .
  • Three markers specific to the Y chromosome were also tested (AMELY at 6.44 mb, DYS19 at 9.13 mb and DYS389 at 13.62 mb) and were not detected in the DNA of the patient.
  • the Y chromosome translocation breakpoint area should be located between these two genes.
  • Applicants used more than fifty Y chromosome-specific PCR markers to walk the chromosome (Fig. 3) . Because of the high degree of homology (99%) between YpIl.2 and Xq21.31, numerous BACs sequences located between SRY (2.30 mb) and AMELY (6.44 mb) genes were compared using BLAST (NCBI) and/or BLAT (Genome Browser, UCSC) to the X chromosome sequence. Primers were positioned in rare regions of the Y chromosome that were distinct from the X chromosome. Positive controls (men) and negative controls (women) were also included.
  • This area mainly contains repeat elements, especially long terminal repeats (LTRs) .
  • LTRs long terminal repeats
  • TGIF2LY gene TGF beta- induced transcription Factor 2-like, OMIM 400025
  • OMIM 400025 TGF beta- induced transcription Factor 2-like, OMIM 400025
  • X chromosome the inverse PCR method was used (Ochman et al. , 1988), this method allows determination of an unknown sequence of the genome positioned beside a known sequence.
  • Inverse PCR has been used to sequence breakpoint of translocations or other rearrangements (Forrester et al., 1999; Akasaka et al., 2000) .
  • Inverse PCR was performed on the genomic DNA of the patient, but also on normal men and women. A 2kb DNA fragment specific to the patient (not observed in the control men and women) was obtained and sequenced. This DNA fragment corresponded to the exact Y chromosome breakpoint sequence position established using the Y chromosome specific PCR (4.205 mb) .
  • the complete sequence of the translocation is illustrated in Figure 4.
  • the first 473 nucleotides show 100% homology to the BAC DNA sequence AC012077 that covers the YpIl.2 chromosome. Variations from this sequence are observed starting from position 474, which is an A/G transition, suggesting that the DNA sequence shifted to X chromosome at this position. Thereafter, variations are increasingly frequent confirming the shift of chromosome. At position 967, a complete change in the homology sequence was observed. In fact, there is no more homology between the X chromosome translocation sequence and the Y chromosome.
  • the X chromosome breakpoint sequence was not recognized, using BLAST (GeneBank) or BLAT (Genome Browser) , in the human genome sequence, suggesting that the X chromosome breakpoint of the patient is located in an unknown region (region that has not been sequenced by the Human Genome Project) .
  • BLAST GeneBank
  • BLAT Gene Browser
  • a portion of the PARl region may be duplicated on the derivative chromosome (see figure 5), and thus, the patient may harbour a partial trisomy of the PARl region.
  • the SLE patient clearly shows three copies of each marker until the end of the PARl region.
  • the 12 known genes found from IL3RA to the end of the PARl region are in three copies in the SLE patient (Fig. 6 and Table 3) .
  • Table 3 List of the triplicated genes that are located between IL3RA and the end of the PARl region in the patient's genomic DNA.
  • the IL3RA gene encode for the alpha subunit of the receptor of interleukin 3 which is a pleotrophic growth factor affecting the proliferation and differentiation of hematopoietic stem cells (committed progenitors of several lineages including megakaryocytes and lymphocytes) .
  • interleukin 3 a pleotrophic growth factor affecting the proliferation and differentiation of hematopoietic stem cells (committed progenitors of several lineages including megakaryocytes and lymphocytes) .
  • Fishman et al. (1993) have shown that the serum level of IL-3 determined by ELISA was higher in a cohort of 16 patients with SLE in comparison to healthy controls.
  • the literature does not indicate other relationship between IL3RA and
  • CD99 (OMIM 313470, alias MIC2) encodes a ubiquitous 32 kDa transmembrane protein (Aubrit et al., 1989) which is expressed on all human tissues tested, in particular, highly expressed in cortical thymocytes and T cells. It has been reported that CD99 is a cell surface molecule involved in adhesion processes and apoptosis of T- cell (Jung et al. , 2003; Kim et al., 2003; Sohn et al. , 1998) . It is interesting to note that in lupus, T-cell seem to play a crucial role. Notably, a number of mutant mice have been reported that point to defective T-cell signal transduction as one potential cause of lupus (King et al. , 2004) .
  • Example 4 Analysis of CD99 expression in lymphocytes and in polymorphonuclear cells.
  • CD99 was over expressed in the patient's immune cells
  • gene expression was measured using a gene expression TaqMan assay (ABI, hs00365982_ml) on the patient's white blood cells and was compared to 6 normal individuals (male and female) , .
  • the relative expression of the CD99 gene was considerably over expressed as compared to the other normal individuals.
  • the level of expression is 2 to 3 times higher in the patient.
  • the level of CD99 expression has also been measured in the polymorphonuclear white blood cells of the patient and control individuals.
  • the relative expression of the CD99 gene was considerably over expressed as compared to the other normal individuals ( Figure 8) .
  • CD99 activates a caspase-independent death pathway in T cells.
  • engagement of a distinct epitope of CD99 by a monoclonal antibody induced kinetically faster and more profound death responses as compared with the impact of anti-FAS and TNF-related apoptosis-inducing ligand.
  • CD99 may play a distinct role in T cell biology, especially in T cell apoptosis.
  • Shin et al. (1999) have demonstrated that in cortical thymocytes a reduction of expression of CD99 is associated with thymic enlargement and markedly reduced apoptosis.
  • lymphopenia Based on the fact that the patient has a very low level of lymphocytes (lymphopenia) , it can be hypothesized that over CD99 expression may be linked to his pathological condition. Interestingly, King et al. (2004) have demonstrated recently that lymphopenia generate autoimmunity via the remaining T cell who undergo vigorous compensatory expansion to reconstitute the immune system. Combined with his familial background of autoimmunity, the lymphopenic condition caused by a higher rate of apoptosis is probably a key event in the physiopathology of this SLE patient.
  • Namjou B Nath SK, Kilpatrick J, Kelly JA, Reid J, James JA, Harley JB (2002a) Stratification of pedigrees multiplex for systemic lupus erythematosus and for self-reported rheumatoid arthritis detects a systemic lupus erythematosus susceptibility gene (SLERl) at 5pl5.3.
  • Namjou B Nath SK, Kilpatrick J, Kelly JA, Reid J, Reichlin M, James JA,

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Abstract

Cette invention a pour objet des gènes localisés dans la région pseudoautosomale 1 (PAR1) et leur utilisation dans le traitement, la prévention et le diagnostic de maladies auto-immunes. Plus précisément, cette invention a pour objet l'inhibition de l'expression/activité des gènes localisés dans la région PAR1 et des polypeptides encodés par ces gènes. Cette invention concerne également des procédés de traitement ou de prévention d'une maladie auto-immune, des procédés de diagnostic ou de pronostic d'une maladie auto-immune et des procédés de dépistage d'agents pouvant traiter ou prévenir les maladies auto-immunes. Les utilisations correspondantes et des emballages commerciaux sont prévus. En outre, cette invention concerne également une nouvelle séquence ADN de la région PAR1.
PCT/CA2005/001335 2004-09-01 2005-09-01 Genes de la region pseudoautosomale 1 et leur utilisation dans le diagnostic, le traitement et la prevention de maladies auto-immunes Ceased WO2006024169A1 (fr)

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Cited By (2)

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EP2492353A4 (fr) * 2009-10-23 2013-06-05 Tokyo Metropolitan Inst Medical Science Biomarqueurs servant à prédire l'effet thérapeutique d'un traitement de désensibilisation
WO2016149682A3 (fr) * 2015-03-18 2017-01-26 Memorial Sloan-Kettering Cancer Center Compositions et procédés de ciblage de cd99 dans les malignités lymphoïdes et hématopoïétiques

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WO1999047538A1 (fr) * 1998-03-19 1999-09-23 Human Genome Sciences, Inc. Analogue de chaine gamma commune de recepteur de cytokine
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KREMER E ET AL: "A cytokine receptor gene cluster in the X-Y pseudoautosomal region", BLOOD, vol. 82, 1993, pages 22 - 28 *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2492353A4 (fr) * 2009-10-23 2013-06-05 Tokyo Metropolitan Inst Medical Science Biomarqueurs servant à prédire l'effet thérapeutique d'un traitement de désensibilisation
WO2016149682A3 (fr) * 2015-03-18 2017-01-26 Memorial Sloan-Kettering Cancer Center Compositions et procédés de ciblage de cd99 dans les malignités lymphoïdes et hématopoïétiques

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