WO2006024169A1

WO2006024169A1 - Genes of the pseudoautosomal region 1 and their use in the diagnosis, treatment and prevention of autoimmune diseases

Info

Publication number: WO2006024169A1
Application number: PCT/CA2005/001335
Authority: WO
Inventors: Pierre Chagnon; Lambert Busque
Original assignee: HEMAX GENOME Inc
Current assignee: HEMAX GENOME Inc
Priority date: 2004-09-01
Filing date: 2005-09-01
Publication date: 2006-03-09
Anticipated expiration: 2007-03-01

Abstract

The present invention relates to genes located in of the pseudoautosomal region 1 (PAR1) and their use in the treatment, prevention and diagnosis of autoimmune diseases. More specifically, the present invention relates to the inhibition of the expression/activity of genes located in the PART region and the polypeptides they encode. Methods of treating or preventing an autoimmune disease, methods of diagnosticating or prognosticating an autoimmune disease and methods of screening for agents capable of treating or preventing autoimmune diseases are also provided. Corresponding uses and commercial packages are described. In addition, a novel DNA sequence of the PAR1 region is also disclosed.

Description

GENES OF THE PSEUDOAUTOSOMAL REGION 1 AND THEIR USE IN THE DIAGNOSIS, TREATMENT AND PREVENTION OF AUTOIMMUNE DISEASES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of United States Provisional Patent Application No. 60/606,137, filed September 1, 2004, which is incorporated herein by reference-.

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION The exact etiology of autoimmunity or autoimmune diseases remains to be elucidated. It has been proposed that environmental factors, hormonal factors, diet and exposure to various substances (sun rays, organic solvents, drugs, infectious organisms) may contribute to the onset and/or the progression of such diseases. A link between genes and autoimmunity has also been proposed since autoimmune diseases tend to aggregate in certain families (Roitt and Delves, 2001) .

Systemic Lupus Erythematosus (SLE, OMIM 152700) is a chronic autoimmune disease characterized by a multisytem organ involvement (skin, joints, kidneys and serosal membranes) and production of auto- antibodies directed against various cellular components. 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

(Namjou et al., 2002b), lq21-22 (Harley and Kelly, 2002), 16ql3 (Gaffney et al., 1998), 17pl3 (Nath et al. , 2001), and 4pl6 (Nath et al. , 2002) .

It is of interest to note that for most of those studies, markers on the X chromosome were not included or analysed because of the specific pattern of transmission of this non autosomal chromosome. If there is a susceptibility gene for SLE on this chromosome it could have been missed.

One of the most striking and intriguing particularity of SLE is its female predominance (Lawrence et al., 1998) . 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. However, 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. Furthermore, the suspected increased incidence of SLE in male patients with XXY Klinefelter syndrome and female with 47XXX supports the role of the X- chromosome in the pathogenesis of SLE (Lahita, 2000; Simko et al. , 2000; Gilliland and Stashower, 2000, Lenoble and Kaplan, 1987; Kurosawa et al. , 1991) . However no specific gene have been mapped or implicated so far.

There is thus a need for the identification of new genes implicated in autoimmunity, especially in SLE. These new genetic targets could serve for the prevention or treatment of autoimmune diseases as well as for the diagnosis and prognosis of such diseases.

SUMMARY OF THE INVENTION

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. According to one aspect of the present invention, there is provided a method of treating or preventing an autoimmune disease in a subject. The method 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. In an embodiment, the gene is selected from the group consisting of CD99, IL3RA, SLC25A6, AK123701, FLJ13330, ASMTL, P2RY8, DXYS155E, ASMT, ZBEDl, BC019893 and AK096998. When the gene is CD99, it 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. When 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. In another embodiment, 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 connective tissue disease. When the disease 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. In an another embodiment, 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. When the immune cell is a lymphocyte, the lymphocyte may be selected from the group consisting of a T lymphocyte and a B lymphocyte.

In a further embodiment, the method can be applied to a mammal, and further to a human.

According to another aspect of the present invention, there is provided use of 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. In an embodiment, the gene may be selected from the group consisting of CD99, IL3RA, SLC25A6, AK123701, FLJ13330, ASMTL, P2RY8, DXYS155E, ASMT, ZBEDl, BC019893 and AK096998. In another embodiment, 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 connective tissue disease. When the disease 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. In a further embodiment the agent may be adapted for administration to an immune cell. In such embodiment, 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. When the immune cell is a lymphocyte, the lymphocyte may be selected from the group consisting of a T lymphocyte and a B lymphocyte. In an embodiment, the use can performed in a mammal, and further, in a human.

According to still another aspect of the present invention, there is provided 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. In an embodiment, the gene may be selected from the group consisting of CD99, IL3RA, SLC25A6, AK123701, FLJ13330, ASMTL, P2RY8, DXYS155E, ASMT, ZBEDl, BC019893 and AK096998. In another embodiment, 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 mixed connective tissue disease. When the disease 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. In yet another embodiment, the agent may be adapted for administration to an immune cell. In such embodiment, 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. When the immune cell is a lymphocyte, the lymphocyte may be selected from the group consisting of a T lymphocyte and a B lymphocyte.

According to yet another aspect of the present invention, there is provided an isolated nucleic acid comprising the sequence of SEQ ID NO: 29.

According to a further aspect of the present invention, there is provided a pharmaceutical composition for use in the treatment or the prevention of an autoimmune disease. In an embodiment, 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. In another embodiment, the gene may be selected from the group consisting of CD99, IL3RA, SLC25A6, AK123701, FLJ13330, ASMTL, P2RY8, DXYS155E, ASMT, ZBEDl, BC019893 and AK096998. In a further embodiment, the composition can be adapted for administration to an immune cell. In such embodiment, 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. When the immune cell is a lymphocyte, the lymphocyte may be selected from the group consisting of a T lymphocyte and a B lymphocyte.

According to yet a further aspect of the present invention, there is provided a method of diagnosing or prognosticating an autoimmune disease in a subject. In an embodiment, the method comprises assessing whether a test parameter is increased with respect to a control parameter. In a further embodiment, 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. In yet another embodiment, an increase in the test parameter is an indication that the subject has an increased likelihood of developing the autoimmune disease. In a further embodiment, the gene is selected from the group consisting of CD99, IL3RA, SLC25A6, AK123701, FLJ13330, ASMTL, P2RY8, DXYS155E, ASMT, ZBEDl, BC019893 and AK096998. In yet another embodiment, 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. In another embodiment, 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 mixed connective tissue disease. When the disease 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. In yet a further embodiment, the 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. In such embodiment, the control subject is a subject at an earlier time or a subject substantially free of said autoimmune disease. In another embodiment, the gene can be selected from the group consisting of CD99, IL3RA, SLC25A6, AK123701, FLJ13330, ASMTL, P2RY8, DXYS155E, ASMT, ZBEDl, BC019893 and AK096998. In yet another embodiment, the method may also comprise performing a polymerase chain reaction. In yet a further embodiment, the test parameter and the control parameter is the copy number of said gene. In such embodiment, the polymerase chain reaction is a real-time quantitative polymerase chain reaction. In another embodiment, the test parameter and the control parameter is the level of expression of said gene. In such embodiment, the polymerase chain reaction is a reverse- transcriptase polymerase chain reaction. According to still a further aspect of the present invention, there is provided 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. In an embodiment, the means is a set of primers selected from the group consisting of:

(i) 5'-CCTCTGTGGGCGTCCGTTCAT-S' (SEQ ID NO: 54) and 5'- CGGGTTCTGCTTGTCTCCATA-3' (SEQ ID NO: 55);

(ii) 5'-TGCCGCAGTGAGAGGAGAAAC-S' (SEQ ID NO: 56) and 5'- GACAGGATGACTGGGCACCAC-3' (SEQ ID NO: 57);

(iii) 5'-GCAATGCCATTATTTATGTCTCA-S' (SEQ ID NO: 58) and 5'- CTCCTGTGGGCTCCTGGGAACTC-3' (SEQ ID NO: 59);

(iv) 5'-GCTGAGTCTTCTGCCCTTCTGA-S' (SEQ ID NO: 60) and 5'- CCCTACCCGAAGCCTGTTTCTC-3' (SEQ ID NO: 61); (v) 5'-AATAGTGGCCGAGGAGGGTGAC-S' (SEQ ID NO: 62) and 5'- ACCTGGCAAGTGCTCAGAGTTC-3' (SEQ ID NO: 63);

(vi) 5'-AGGAGGTTCTATTTCGGGTGTC-S' (SEQ ID NO: 64) and 5'- CTTGGGCTGGTGGCAGAGGA-3' (SEQ ID NO: 65);

(vii) 5'-TACTGCTCCCATCGCTGGTGTC-S' (SEQ ID NO: 66) and 5'- GCCTCATTTCACTTTCACATCC-S' (SEQ ID NO: 67);

(viii) 5'-ACTCTCCAGCGGTTCTCAAAG-S' (SEQ ID NO: 68) and 5'GCTAAAGCCGTGGTATCACAGS' (SEQ ID NO: 69);

(ix) 5'-CATCCCGAATGACTTCCTGTT-S' (SEQ ID NO: 70) and 5'- CCCGTTGACGCTCCAGACCTC-3' (SEQ ID NO: 71); (x) 5'-AGAGCAGCCGTCTCCCATCCT-S' (SEQ ID NO: 72) and 5'- ACCCACCGCACTCACCTTCAT-3' (SEQ ID NO: 73);

(xi) 5'-TGCCCACCTGCTCTGTAGAATC-S' (SEQ ID NO: 74) and 5'- TCATCACGCCCTGTCCCTCA-3' (SEQ ID NO: 75);

(xii) 5'-GCACTTAGACATTAGCCCGAGAA-S' (SEQ ID NO: 76) and 5'- CACGGCAGGAGTCTCAGTTTCAG-3' (SEQ ID NO: 77); and (xiii) 5'-TTTACACGCTCAAGGCCAGTC-S' (SEQ ID NO: 78) and 5'- CATCCACGTTGGCAAACTGCT-3' (SEQ ID NO: 79) .

According to another aspect of the present invention, there is provided a method of screening for an agent capable of treating or preventing an autoimmune disease in a subject. In an embodiment, 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. In a further embodiment, the inhibition is indicative that the agent is capable of treating or preventing the autoimmune disease. In an another embodiment, the gene may be selected from the group consisting of CD99, IL3RA, SLC25A6, AK123701, FLJ13330, ASMTL, P2RY8, DXYS155E, ASMT, ZBEDl, BC019893 and AK096998. In a further embodiment, 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 connective tissue disease. When the disease 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. In yet another embodiment, the assessment is performed in an immune cell. In such embodiment, 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. When the immune cell is a lymphocyte, the lymphocyte may be selected from the group consisting of a T lymphocyte and a B lymphocyte. In yet a further embodiment, the method also comprises performing a polymerase chain reaction. In such embodiment, the polymerase chain reaction may be a reverse-transcriptase polymerase chain reaction.

According to yet another aspect of the present invention, there is provided 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. In an embodiment, the gene is selected from the group consisting of CD99, IL3RA, SLC25A6, AK123701, FLJ13330, ASMTL, P2RY8, DXYS155E, ASMT, ZBEDl, BC019893 and AK096998. In another embodiment, 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 connective tissue disease. When the disease 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. In a further embodiment, the instructions specify the use of an immune cell. In such embodiment, 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. When the immune cell is a lymphocyte, the lymphocyte may be selected from the group consisting of a T lymphocyte and a B lymphocyte. In yet another embodiment, the instructions specify the use of a polymerase chain reaction. In such embodiment, the polymerase chain reaction may be a reverse-transcriptase polymerase chain reaction. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 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. 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) .

Figure 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) .

Figure 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) indicated that 93% of the sequence consists of repeated elements and that the position between 1 and 966 is made up of LTR/ERVK repeat sequences. 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 9. Human CD99 coding sequence (SEQ ID NO: 3, Accession Number

NM_002414) and polypeptide sequence (SEQ ID NO: 4, Accession Number NPJD02405) .

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 19. Human ZBEDl coding sequence (SEQ ID NO: 23, Accession Number NM_004729) and polypeptide sequence (SEQ ID NO: 24, Accession Number ^' NP_004720) .

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

Number AK096998) and polypeptide sequence (SEQ ID NO: 28, Accession Number BACO4924) .

Figure 22. Novel sequence from the X chromosome breakpoint, nucleotides 967 to 1668 from Figure 4 (SEQ ID NO: 29) .

DETAILED DESCRIPTION OF THE INVENTION

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. In a first aspect, the invention relates to a method of treating or preventing an autoimmune disease. What is meant herein by "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) . The terms "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. These 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.

In order to treat or prevent an autoimmune disease, 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.

As used herein, the terms "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. The

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 BC019893) and AK096998 coding sequence (SEQ ID NO: 27, Accession Number AK096998) . The CD99 gene is present in various organism, such as the green African monkey (nucleotide accession number U82166.1 and protein accession number AAB93833) . A person of ordinary skill in the art recognizes that sequence of the above-mentioned genes may vary from one individual to another (e.g. allelic variation). As such, 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. For example, table 1 below 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) . Two nucleic acid sequences are considered substantially identical if, when optimally aligned (with gaps permitted) , they share at least about 50% sequence similarity or identity, or if the sequences share defined functional motifs. In alternative embodiments, sequence similarity in optimally aligned substantially identical sequences may be at least 60%, 70%, 75%, 80%, 85%, 90% or 95%. As used herein, 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. Natl. Acad. Sci. USA 85: 2444, and the computerised implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI, U.S.A.). Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al. ,

1990, J. MoI. Biol. 215:403-10 (using the published default settings) . Software for performing BLAST analysis may be available through the National Center for Biotechnology Information (through the internet at http: //www.ncbi.nlm.nih.qov/) . 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. 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

89: 10915-10919) alignments (B) of 50, expectation (E) of 10 (or 1 or 0.1 or 0.01 or 0.001 or 0.0001), M=5, N=4, and a comparison of both strands. One measure of the statistical similarity between two sequences using the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. In alternative embodiments of the invention, 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.

An alternative indication that two nucleic acid sequences are substantially complementary is that the two sequences hybridize to each other under moderately stringent, or preferably stringent, conditions. Hybridisation to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHPO₄, 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) . Alternatively, hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHPO₄, 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) . Generally, 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.

The methods described herein relate to autoimmunity and autoimmune diseases. 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. Further, "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 connective tissue disease. In addition, lupus includes, but is not limited to, systemic lupus erythematosus, discoid lupus erythematosus, subacute cutaneous lupus erythematosus, drug-induced lupus and neonatal lupus.

In an embodiment, 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. 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) and/or gold salts. Therefore, in alternative embodiments, 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. Examples of therapeutic antisense oligonucleotide applications, incorporated herein by reference, 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. Pat. No. 5,194,428, issued Mar. 16, 1993; U.S. Pat. No. 4,806,463, issued Feb. 21, 1989; U.S. Pat. No. 5,286,717 issued Feb. 15, 1994; U.S. Pat. No. 5,276,019 and U.S. Pat. No. 5,264,423; BioWorld Today, Apr. 29, 1994, p. 3. Preferably, in antisense molecules, there is a sufficient degree of complementarity to the transcripts of genes of the PARl region to avoid non-specific binding of the antisense molecule to non-target sequences under conditions in which specific binding is desired, such as under physiological conditions in the case of in vivo assays or therapeutic treatment or, in the case of in vitro assays, under conditions in which the assays are conducted. 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. A method of screening for antisense and ribozyme nucleic acids that may be used to provide such molecules as inhibitors of the invention is disclosed in U.S. Patent No. 5,932,435 (which is incorporated herein by reference) .

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 CH₂--NH—O—CH₂, CH₂—N(CH₃) —0--CH₂ (known as methylene (methylimino) or MMI backbone), CH₂—O—N(CH₃) —CH₂, CH₂—N(CH₃) —N(CH₃) —CH₂ and O—N(CH₃) —CH₂ —CH₂ backbones (where phosphodiester is O—P—0—CH₂) . Oligonucleotides having morpholino backbone structures may also be used (U.S. Pat. No. 5,034,506) . In alternative embodiments, 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. Thus, purines and pyrimidines other than those normally found in nature may be used. Similarly, 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. Some specific examples of modifications at the 2' position of sugar moieties which are useful in the present invention are OH, SH, SCH₃, F, OCN, 0(CH₂)_n NH₂ or 0 (CH₂)„ CH₃ where n is from 1 to about 10; C₁ to Ci₀ lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF₃ ; OCF₃ ; O-, S-, or N-alkyl; 0-, S-, or N-alkenyl; SOCH₃ ; SO₂ CH₃; ONO₂ ; NO₂ ; N₃; NH₂; 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 pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. One or more pentofuranosyl groups may be replaced by another sugar, by a sugar mimic such as cyclobutyl or by another moiety which takes the place of the sugar.

In some embodiments, the antisense oligonucleotides in accordance with this invention may comprise from about 5 to about 100 nucleotide units. As will be appreciated, 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.

In a further embodiment, 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. As such, 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. Such a system may be used for functional studies of the product, as well as to treat disorders related to the activity of such a product. 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) .

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. Alternatively, 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. vector encoding precursor(s) , etc.) of such an siRNA or siRNA-like molecule. A siRNA may then associate with other intracellular components to form an 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. Thus the target mRNA is cleaved and the level of protein product it encodes is reduced.

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. coli RNase III) in vitro to a size conducive to RNAi. The sense and antisense transcripts are combined to form an 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. Various vector-based methods are described in for example Brummelkamp et al.

(2002), Lee et al. (2002), Miyagashi and Taira (2002), Paddison et al. (2002) Paul et al. (2002) Sui et al. (2002) and Yu et al. (2002) . Various methods for introducing such vectors into cells, either in vitro or in vivo (e.g. gene therapy) are known in the art.

Accordingly, in an embodiment expression of a nucleic acid encoding a polypeptide of interest, or a fragment thereof, may be inhibited by introducing into or generating within a cell an siRNA or siRNA-like molecule corresponding to a nucleic acid encoding a polypeptide of interest, or a fragment thereof, or to an nucleic acid homologous thereto. "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. In various embodiments 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. In an embodiment, the siRNA or siRNA-like molecule is less than about 30 nucleotides in length. In a further embodiment, the siRNA or siRNA-like molecule is about 21-23 nucleotides in length. In an embodiment, siRNA or siRNA- like molecule comprises a 19-21 bp duplex portion, each strand having a 2 nucleotide 3' overhang. In embodiments, 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. In embodiments, 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) . In an embodiment, 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.

As such, the methods and agents described herein may be adapted to act on immune cells. These cells include, but are not limited to, lymphocyte (e.g. B and T lymphocytes), dendritic cell, monocyte, macrophage, neutrophil (e.g. polymorphonuclear cell or PMN), eosinophil, basophil and mast cell. Many subjects can benefit from the invention described herein. In an embodiment, the subject is a mammal, and further, a human.

In a further aspect, 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. As mentioned above, 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.

In another aspect, the invention relates to an isolated nucleic acid comprising the following sequence (nucleotides 967 to 1668 from Figure 4) :

CTGAGGGAAGAGAGAGACCCTCTCATATTGTTTTATGTTGTTTTATACTCATTACCTGTTTTAAGAAAACAG

ATTGTGCATTGGAGAGCTGGGATTTTAAGGCAGTAGCTTCCCGATGCTCCCAGCTGAATAAAGCCCTTCCTT

TGAΆAAAGTTGAGAAGGCAGATACTAAAGAGAAGAAACCTGAAGCCAAGAΆGGC (SEQ ID NO: 29) .

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) . In addition, 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.) . In another aspect of the invention, an isolated nucleic acid, or homolog, fragment or variant thereof, may further be incorporated into a recombinant expression vector. In an embodiment, 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. For instance, a promoter is operably-linked to a coding sequence if the promoter affects the transcription or expression of the coding sequences. Generally, operably-linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame. However, since for example 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 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

Molecular Cloning: A Laboratory Manual. A variety of strategies are available for ligating fragments of DNA, the choice of which depends on the nature of the termini of the DNA fragments and can be readily determined by persons skilled in the art. The vectors of the present invention may also contain other sequence elements to facilitate vector propagation and selection in bacteria and host cells. In addition, 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. Accordingly, the invention also provides host cells containing the recombinant expression vectors of the invention. The terms "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. The terms "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.

In yet another aspect, 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.

In one aspect, 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. Typically, 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. Thus, for example, 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,

HPLC, etc .

In various embodiments, 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. In one embodiment, 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. For any particular subject, 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.

As used herein "pharmaceutically acceptable carrier" or "excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In one embodiment, the carrier is suitable for parenteral administration. Alternatively, 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.

Therapeutic 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. In many cases, it will be preferable to include 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. Moreover, 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. Generally, 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. In the case of sterile powders for the preparation of sterile injectable solutions, 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. In accordance with an alternative aspect of the invention, an agent may be formulated with one or more additional compounds that enhance the solubility of the agent.

Different diagnostic methods were developed and are still used to assess autoimmune diseases. 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. Certains specific auto-antibodies are linked to a specific disease whereas others are linked to a general auto-immune response.

For example, in SLE, many different 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) . 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.

In addition to auto-antibodies, routine laboratory tests can also be performed to assess the presence or severity of an autoimmune disease. For example, blood count, urinalysis, blood chemistries, erythrocyte sedimentation rate and complement can be used to diagnose SLE or to measure the severity of the disease.

Further, the presence of certains symptoms, alone or in combination, can also help a physician to diagnose an autoimmune disease. For example, 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.

In a further aspect, 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. As used herein, an "increased likelihood of developing an autoimmune disease" is defined an increased risk, for a subject, of developing an autoimmune disease or that he may continue experiencing an autoimmune disease. The 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. In an embodiment, the gene is a gene located in a PARl region and the protein is a protein encoded by such gene. In yet a further embodiment, 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. As such, 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. cytochrome C release assay), cathepsin and calpain assays, etc. On the other hand, 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. For example, 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. Since IL3RA can also activate STAT5, by indirectly inducing its phosphorylation (e.g. tyrosine phosphorylation), the level of phosphorylation of STAT5 can also be determined to assess ILR3A activity. 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.

In the diagnostic/prognostic method described herein, 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. Alternatively, 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. In an embodiment, in order to assess the alteration (e.g. increase) in the test parameter, 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

PARl region in a control subject and the activity of a protein encoded by a gene located in a PARl region in a control subject. The gene located in a PARl region can be selected from the genes described herein. In addition, the value of the control parameter can be measured with the techniques described herein. 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) . In addition, 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:

(i) 5'-CCTCTGTGGGCGTCCGTTCAT-S' (SEQ ID NO: 54) and 5'-CGGGTTCTGCTTGTCTCCATA-S' (SEQ ID NO: 55); (ii) 5'-TGCCGCAGTGAGAGGAGAAAC-S' (SEQ ID NO: 56) and 5'-GACAGGATGACTGGGCACCAC-S' (SEQ ID NO: 57); (iϋ) 5'-GCAATGCCATTATTTATGTCTCA-S' (SEQ ID NO: 58) and 5'- CTCCTGTGGGCTCCTGGGAACTC-3' (SEQ ID NO: 59);

(iv) 5'-GCTGAGTCTTCTGCCCTTCTGA-S' (SEQ ID NO: 60) and 5'- CCCTACCCGAAGCCTGTTTCTC-3' (SEQ ID NO: 61);

(v) 5'-AATAGTGGCCGAGGAGGGTGAC-S' (SEQ ID NO: 62) and 5'- ACCTGGCAAGTGCTCAGAGTTC-3' (SEQ ID NO: 63);

(vii) 5'-TACTGCTCCCATCGCTGGTGTC-S' (SEQ ID NO: 66) and 5'- GCCTCATTTCACTTTCACATCC-3' (SEQ ID NO: 67); (viii) 5'-ACTCTCCAGCGGTTCTCAAAG-S' (SEQ ID NO: 68) and 5'GCTAAAGCCGTGGTATCACAGS' (SEQ ID NO: 69);

(ix) 5'-CATCCCGAATGACTTCCTGTT-S' (SEQ ID NO: 70) and 5'- CCCGTTGACGCTCCAGACCTC-3' (SEQ ID NO: 71);

(x) 5'-AGAGCAGCCGTCTCCCATCCT-S' (SEQ ID NO: 72) and 5'- ACCCACCGCACTCACCTTCAT-3' (SEQ ID NO: 73);

(xii) 5'-GCACTTAGACATTAGCCCGAGAA-S' (SEQ ID NO: 76) and 5'- CACGGCAGGAGTCTCAGTTTCAG-3' (SEQ ID NO: 77); and (xiii) 5'-TTTACACGCTCAAGGCCAGTC-S' (SEQ ID NO: 78) and 5'-

CATCCACGTTGGCAAACTGCT-3' (SEQ ID NO: 79) .

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. In order to detect such a translocation, 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.

In another embodiment, to detect such triplication, a PCR could also be performed with the subject's genomic DNA using primers located on either side of the translocation. In a further embodiment, 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) .

In another embodiment, multiple sets of primers (e.g. at least two) 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. In an example where both sets of primers are specific to the Y chromosome, 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. In this particular example, the first of primers that can be used may have the following sequences:

5'-ATTTGGGGTCTTAGATTTATGG-S' (SEQ ID NO: 36) and 5'-GTCCTGCACAGCCCTAGATCCC-S' (SEQ ID NO: 37); and 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) .

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 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. In an embodiment, the above-mentioned primers can serves as detection means. In yet a further aspect, 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. Such inhibition being indicative of that the agent is capable of treating or preventing an autoimmune disease. The screening can be performed, for example, in an immune cell. Such 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.

Alternatively, the screening can be performed in a transgenic animal overexpression a gene located in a PARl region.

In another embodiment, 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) . 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. Such a method may comprise assaying gene expression in the presence versus the absence of a test agent. Such 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. For instance, a promoter is operably- linked to a coding sequence if the promoter affects the transcription or expression of the coding sequences. Generally, operably-linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame. However, since for example 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. In certain embodiments, 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. A variety of such detection means may be used, including but not limited to one or a combination of the following: radiolabelling (e.g. ³²P), antibody-based detection, fluorescence, chemiluminescence, spectroscopic methods (e.g. generation of a product with altered spectroscopic properties), various reporter enzymes or proteins (e.g. horseradish peroxidase, green fluorescent protein), specific binding reagents (e.g. biotin/ (strept)avidin) , and others. 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. Such 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. Such 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. Such 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. In an embodiment, the above- mentioned primers can serves as detection means.

Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. In the claims, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including, but not limited to". The following examples are illustrative of various aspects of the invention, and do not limit the broad aspects of the invention as disclosed herein. EXAMPLES

Example 1 — Material and methods

Cell lines. Lymphocytes from the patient (see below) 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.

Karyotyping. Conventional cytogenetic analysis has been performed on culture cells by standard techniques and evaluated by Giemsa-Trypsin-Giemsa (GTG) banding at about the 400 band level according to the International System for Human Cytogenetic Nomenclature (BCLQ) . The human Spectral Karyotyping (SKY) is designed to enable simultaneous visualization of all human chromosomes. SKY was performed on a normal patient metaphase chromosome slide preparation to assure that the patient did not present an abnormality on other chromosome. The distinction between the dyes was performed with SkyVision™ from AEPLIED SPECTRAL IMAGING.

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 CytoVision™ System software (Applied Imaging, Santa Clara, CA) . Available commercial fluorescence probes were used: double STS-CEP X (LSI (locus specific identifier DNA probe) - (chromosome enumeration DNA FISH probe)) which correspond to region Xp22.3 (STS) and Xpll.l-qll.l (CEP X) ; single CEP X; SRY (LSI-sex determining region-Y) (Vysis, LSI® probes, Downers Grove, IL) .

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. Because of the high degree of homology (99%) between the YpIl.2 and Xq21.31 chromosome, numerous Y bacterial artifical chromosomes (BACs) sequences located between SRY and AMELY genes with the X chromosome were compared using BLAST (NCBI) and/or BLAT (Genome Browser, UCSC) (e.g. AC006040, AC074181, AC006157, AC006032, AC019058, AC010094, AC010106, AC024703, AC012077, AC010142, AC010129, AC010977, AC006335, AC010154, AC010144, AC010728, AC013412, AC011297 and AC012068) . Primers were designed and selected to be specific only for the Y chromosome. To detect the exact position of the breakpoint on the Y chromosome, more than 50 markers have been designed. These markers were tested with PCR on DNA obtained from the patient (Blood and cultured cells), normal men (positive controls) and normal DNA women (negative controls) . 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 50⁰C, 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 50⁰C, chromosome Y position 4.197 Mb; D: forward, 5'-ATTTGGGGTCTTAGATTTATGG-S' (SEQ ID NO: 36) and reverse, 5'- GTCCTGCACAGCCCTAGATCCC-3' (SEQ ID NO: 37), annealing temperature 53°C, chromosome Y position 4.205 Mb; E: forward, 5'-TGAACCACACTTGCATCACTG- 3' (SEQ ID NO: 38) and reverse, 5'-AAAAGTCTTCCAGTCCATAAC-S' (SEQ ID NO: 39), annealing temperature 52°C, chromosome Y position 4.207 Mb; F: forward, 5'-ATAGTTTGAAGCCATGTAGGA-S' (SEQ ID NO: 40) and reverse, 5'- TTGCTAAAGATACTGGCCTCC-3' (SEQ ID NO: 41), annealing temperature 53°C, chromosome Y position 4.216 Mb; G: forward, 5'-TGTAGACATCACCATTATCCA- 3' (SEQ ID NO: 42) and reverse, 5'-TCTTCTATGTATGAACTTT-S' (SEQ ID NO: 43), annealing temperature 50⁰C, chromosome Y position 4.232 Mb; H: forward, 5'-CCTTGTTCTGATCCACGACC-S' (SEQ ID NO: 44) and reverse, 5'- CAAGCTCTGGAGGTTGTGGG-3' (SEQ ID NO: 45), annealing temperature 54°C, chromosome Y position 4.241 Mb. Inverse PCR. Genomic DNA samples (2 μg) , from a normal individuals and the patient, were digested overnight (0/N) at 37°C with 30 U of Kpnϊ and 2 μl of the appropriate buffer (NEB) in a total volume of 20 μl. 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 μl of ddH₂O. 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 μl of the appropriate buffer (NEB) in a total volume of 500 μl. Extra ATP was added to a final concentration of 1 mM. The reactions were incubated at 16^CC 0/N. The ligation products -were ethanol precipitated and resuspended in 15 μl of ddH₂O. Two rounds of PCR were conducted using nested primers for the second round. All the PCR reactions were done in a total volume of 50 μl, with 2.5 U of Taq polymerase, the appropriate buffer (Sigma) and final concentrations of 0.45 μM of each primer, 1.5 mM of MgCl₂, 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 50⁰C and 3 min 30 sec at 72⁰C, and a final elongation step of 7 min at 72⁰C. For the first round, 2 μl of the purified ligation products were used with primers 5'-ATCAAAATGGCAAACAGTCA-S' (SEQ ID NO: 46) and 5'-ATTTGGGGTCTTAGATTTATGG-S' (SEQ ID NO: 47) . For the second round, 2 μl of the first-round PCR products served as templates with nested primers 5'-GTTATATACCCCCTTCTCCC-S' (SEQ ID NO: 48) and 5'- GGGATCTAGGGCTGTGCAGGAC-3' (SEQ ID NO: 49) . All PCR products were visualised on a 1.5 % agarose gel by migrating 10 μl of each reaction.

Sequencing of the breakpoint. 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) . This amplicon was directly sequence using the same PCR primer and also by using nested sequencing primer 5'- CTGTCTGCTGCCTGCCCCTGG-3' (SEQ ID NO: 52) and 5'-TCAGACACCAAGCTGTAGAAG-S' (SEQ ID NO: 53) and was purified by the Millipore multiscreen-PCR 96- well filtration system. PCR products were sequenced using Big Dye Terminator Cycle Sequencing Kit v.3.1 (Applied Biosystems) according to the manufacturer's instructions. Sequence products were analysed on an ABI 3100 sequencer. Isolation of RNA and cDNA synthesis. Total RNA was isolated from MLL (myeloid lymphocyte layer) using TriPure™ Isolation Reagent (Roche Diagnostics, USA) . RNA was treated with Dnase I (Sigma-Aldrich, USA) before cDNA synthesis. Reverse transcription of total RNA was performed using Tagman™ RT reagents (Applied Biosystems, CA, USA) using random hexamers.

Comparative gene expression. The relative expression of the human CD99 gene (SEQ ID NO: 3, Accession number NM_002414) was determined for the patient and six normal individuals (male and female) . The relative quantification of this gene was performed using real-time PCR on an ABI Prism 7000 Sequence Detection System using the standard curve method, as described in the Applied Biosystems User Bulletin No.2 (P/N 4303859B) . Briefly, the target gene was quantified and normalized to an endogenous control gene (human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or human cyclophilin A (CYC) ) , using standard curves for both the target and endogenous genes. The normalized target gene value of the patient was then compared to the normalized values of each normal individual. PCR reactions were carried out on 25-50 ng of cDNA using Taqman™ Universal Master Mix with Assays-on-Demand™ Gene

Expression Products (Applied Biosystems, USA) in a total volume of 25 μl. 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. Thirteen markers covering the PARl region and other X chromosome- specific regions were analyzed using an assay adapted from Laurendeau et al. (1999) . Briefly, the starting copy number of a target gene in a DNA sample was determined by real-time PCR and normalized against the starting copy number of a reference disomic gene (marker 766) . The starting gene copy numbers are inferred from a relative standard curve. This normalized gene dose ratio (N) :

N = starting copy number of target gene starting copy number of reference gene (766) permits determination of the gene copy number in the genome. For instance, a ratio of N=I indicates that the target gene is found in 2 copies in the genome, while N=I.5 implies 3 copies. Hemizygosity is expected to yield a ratio of N=O.5. 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 SYBR™ Green PCR Master Mix (Applied Biosystems, USA) in a total volume of 25 μl. The final concentration of 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 95¹C 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:

Table 2 . Characteristics of the primers used for the real-time quantitative PCR

Example 2 - Case study

A boy was diagnosed with systemic lupus erythematosus (SLE) in 1992 at the age of 6 years. His disease has been characterized by severe rash, severe mucositis, arthritis and focal proliferative glomerulonephritis. His autoantibody profile has included positive antinuclear antibodies (ANA), anti-DNA and anti-smooth muscle (Sm) antibodies. During the course of his disease he also developed elevated liver enzymes and severe, protracted thrombotic thrombocytopenic purpura (TTP) . His disease course has been further complicated by osteoporosis with vertebral fractures and growth failure.

In terms of pubertal development, he has primary hypogonadism with partial Leydig cell failure, suboptimal testosterone secretion and elevated levels of both LH and FSH. He has low testicular volumes with failure of seminiferous tubule development. GH testing has been normal. He has been treated with intramuscular injections of testosterone. In terms of the family history, his mother was diagnosed with Idiopathic Thrombocytopenic Purpura (ITP) at the age of 15 years and was treated with corticosteroids and subsequently had a splenectomy. A maternal aunt also had ITP treated with corticosteroids followed by splenectomy. Apparently both his mother and maternal aunt had positive ANAs.

Treatment of the patient's disease has included corticosteroids, hydroxychloroquine, methotrexate, intravenous pulse cyclophosphamide, azathioprine and plasmapheresis. In addition the patient has required treatment for hypertension and has received pamidronate for osteoporosis.

Example 3 - Genetic analysis

Karyotype analysis of the patient indicated 46 chromosomes with one normal X chromosome and a derivative X;Y translocation chromosome with breakpoint at Xp22.3 and YpIl.2. 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). Spectral karyotyping Skypaint analysis confirmed the Xp22.33/Ypll.2 translocation and did not detect any other chromosome abnormalities (Fig. 1) .

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/) . Two copies of the X chromosome gene STS (steroid sulfatase microsomal, arylsulfatase C, isozyme S) were also detected by FISH (data not shown) , indicating that at this position (6.60 mb according to the July 2003 assembly of the UCSC Genome Browser, http://genome.ucsc.edu/), the patient still possesses his two X chromosomes. Thus, the translocation is located upstream of this position. X inactivation study was performed on DNA extracted from peripheral white blood cells by analysis the polymorphic CAG repeat in the androgen receptor (HUMARA) gene by a modified fluorescence method described by Busque et al (1996) . The X-inactivation studies showed an almost normal random (65% versus 35%) inactivation pattern. This indicated that the derivative X chromosome harbouring the translocation should not be deleterious, because a certain percentage the cells have this X chromosome active. If the translocation was deleterious, a strong skewing in the X inactivation pattern should have been observed because initial cells harbouring the translocation would have been eliminated. To further help in positioning the translocation breakpoint,

54 highly polymorphic 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) . All markers upstream being homozygote, the X chromosome breakpoint has to be located between the telomere and this position, probably in the PARl region. In addition, 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. Considering the presence of SRY gene (detected by FISH (2.30 mb) and the loss of AMELY gene (6.44 mb) on the translocated chromosome, the Y chromosome translocation breakpoint area should be located between these two genes.

In order to determine the exact breakpoint position on the Y chromosome, 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. All markers located between position 2.29 mb and 3.98 mb could be amplified in both the patient and positive control DNAs, but not in women. However, all markers located between 4.22 mb and 8.60 mb were not detected in the patient, but were present in men. Intensive analysis of the Y chromosome region between 3.98 mb and 4.22 mb allowed to determine the exact position of the breakpoint, which is located inside the BAC DNA sequence AC012077 between markers D and E (Fig. 3) . The patient is harbouring marker D, but not E. These two markers are located at positions 4.205 and 4.207 mb on the Y chromosome, in a small area of 2130 bp. The translocation may ^'have occurred in that small region. This area mainly contains repeat elements, especially long terminal repeats (LTRs) . However, downstream on the Y chromosome, the TGIF2LY gene (TGF beta- induced transcription Factor 2-like, OMIM 400025) is observed on the proximal side at 3.15 mb, and on the distal side, the PCDHlIY gene

(Protocadherin 11, Y-linked, OMIM 400022) is also observed at 4.60 mb. This latter gene is absent in the patient. While SRY, RPS4Y, ZFY, and TGIF2LY Y chromosome-specific genes are found in the patient, all other Y chromosome genes are absent. To precisely determine the position of the breakpoint on the

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) . Approximately 1 kb of new sequence was analyzed and found to be the X chromosome part. A pair of DNA primers was designed, one of which was specific to the Y chromosome (5'- AATACTGTCTTCTCCCCAATG-3'SEQ ID NO: 50) and the other specific to the X chromosome (5'-CGTCCTTGTCACCGCCAACTG-B' SEQ ID NO:51) . Using these primers, the proper DNA fragment should only be amplified in individuals harbouring the Xp22.33; YpIl.2 translocation. Indeed, the 2 kb amplicon could only be detected in the patient. Using the same PCR primers and additional nested primers, it was confirmed that the sequence was the same as the one obtained by inverse PCR.

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.

Sequence analysis of the breakpoint sequence with repeatmasker v.2 (http://repeatmasker.genome.washington.edu/) indicated that the abnormal X and Y chromosome interchange occurred between retroviral long-terminal repeats of human endogenous retroviral sequence K. This LTR/HERVK is part of the large family that comprises about 1% of the human genome (Lower et al., 1996) .

Surprisingly, 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) . From the telomere to the GATA164D10 marker, there are three gaps in the X chromosome sequence. Those gaps are located within the PARl region and the translocation breakpoint should be positioned in one of those gaps. As a consequence, 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. In order to determine in which gap the translocation is located and to confirm that some genes are present in three copies in the patient, an assay using SYBRGreen real time quantitative PCR was developed (adapted from Laurendeau et al. (1999)) to map the entire PARl region. Thirteen real-time quantitative PCR reactions covering the PARl region and other X chromosome-specific regions were performed on the patient and control DNA. Figure 6 illustrates that the genes located outside of the PARl region (GYG2 and TM4SF2) are present in 2 copies in our patient and in normal women, while they are found in one copy in normal men. Starting from the telomere, normal individuals (men and women) and the patient possess 2 copies of each marker, until IL3RA gene. From this position, including IL3RA, the SLE patient clearly shows three copies of each marker until the end of the PARl region. Interestingly, there is a gap of 100 kb (position 0,989 to 1,089 mb) just before the IL3RA gene. The translocation may have occurred in that gap. As a consequence, 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.

Interestingly, 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) . In addition, 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

SLE.

On the other hand, 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.

In order to determine if 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) , . As shown in figure 7, the relative expression of the CD99 gene was considerably over expressed as compared to the other normal individuals. In fact, the level of expression is 2 to 3 times higher in the patient. In addition, the level of CD99 expression has also been measured in the polymorphonuclear white blood cells of the patient and control individuals. As mentioned above, the relative expression of the CD99 gene was considerably over expressed as compared to the other normal individuals (Figure 8) .

Interestingly, Pettersen et al. (2001) have demonstrated that CD99 activates a caspase-independent death pathway in T cells. In addition, 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. Thus, CD99 may play a distinct role in T cell biology, especially in T cell apoptosis. Moreover, 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. 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. The uniqueness of the translocation, the rarity of male severe prepubertal SLE and the increased incidence of SLE in Klinefelter and 47XXX females, who show also triplication of the PARl regions, militates in favour of a causative relationship between the partial triplication of the PARl region and the development of autoimmunity (e.g. SLE) in the patient.

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

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Claims

WHAT IS CLAIMED IS:

1. Method of treating or preventing an autoimmune disease in a subject, said method comprising inhibiting the expression of a gene located in a PARl region and/or inhibiting the activity of the polypeptide encoded by said gene.

2. The method according to claim 1, wherein said gene is selected from the group consisting of CD99, IL3RA, SLC25A6, AK123701, FLJ13330, ASMTL, P2RY8, DXYS155E, ASMT, ZBEDl, BC019893 and AK096998.

3. The method according to claim 2, wherein said gene is a CD99 gene.

4. The method according to claim 3, wherein said CD99 gene comprises a nucleotide sequence substantially identical to the nucleotide sequence of SEQ ID NO: 3.

5. The method according to claim -3, wherein said CD99 gene encodes a polypeptide having the amino acid sequence of SEQ ID NO: 4.

6. The method according to claim 2, wherein said gene is a IL3RA gene.

7. The method according to claim 6, wherein said IL3RA gene comprises a nucleotide sequence substantially identical to the nucleotide sequence of SEQ ID NO: 5.

8. The method according to claim 6, wherein said IL3RA gene encodes a polypeptide having the amino acid sequence of SEQ ID NO: 6.

9. The method according to claim 1, wherein said 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 connective tissue disease.

10. The method according to claim 9, wherein said autoimmune disease is lupus.

11. The method according to claim 10, wherein said lupus is selected from the group consisting of systemic lupus erythematosus, discoid lupus erythematosus, subacute cutaneous lupus erythematosus, drug-induced lupus and neonatal lupus.

12. The method according to claim 1, said method comprising administering an agent capable of inhibiting the expression of said gene and/or the activity of said protein.

13. The method according to claim 1, said method comprising inhibiting the expression of a gene located in a PARl region and/or inhibiting the activity of a polypeptide encoded by said gene in an immune cell.

14. The method according to claim 13, wherein said immune cell is 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.

15. The method according to claim 14, wherein said immune cell is a lymphocyte.

16. The method according to claim 15, wherein said lymphocyte is selected from the group consisting of a T lymphocyte and a B lymphocyte.

17. The method according to claim 1, wherein said subject is a mammal.

18. The method according to claim 17, wherein said mammal is a human.

19. Use of an agent capable of inhibiting the expression of a gene located in a PARl region and/or the activity of a polypeptide encoded by said gene for the treatment or prevention of an autoimmune disease in a subject.

20. The use according to claim 19, wherein said gene is selected from the group consisting of CD99, IL3RA, SLC25A6, AK123701, FLJ13330, ASMTL, P2RY8, DXYS155E, ASMT, ZBEDl, BC019893 and AK096998.

21. The use according to claim 19, wherein said 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 connective tissue disease.

22. The use according to claim 21, wherein said autoimmune disease is lupus.

23. The use according to claim 22, wherein said lupus is selected from the group consisting of systemic lupus erythematosus, discoid lupus erythematosus, subacute cutaneous lupus erythematosus, drug-induced lupus and neonatal lupus.

24. The use according to claim 19, wherein said agent is adapted for administration to an immune cell.

25. The use according to claim 24, wherein said immune cell is 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.

26. The use according to claim 25, wherein said immune cell is a lymphocyte.

27. The use according to claim 26, wherein said lymphocyte is selected from the group consisting of a T lymphocyte and a B lymphocyte.

28. The use according to claim 19, wherein said subject is a mammal.

29. The use according to claim 28, wherein said mammal is a human.

30. 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 said gene; and

• instructions for use of said agent in the treatment or prevention an autoimmune disease.

31. The commercial package according to claim 30, wherein said gene is selected from the group consisting of CD99, IL3RA, SLC25A6, AK123701, FLJ13330, ASMTL, P2RY8, DXYS155E, ASMT, ZBEDl, BC019893 and AK096998.

32. The commercial package according to claim 30, wherein said 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 connective tissue disease.

33. The commercial package according to claim 32, wherein said autoimmune disease is lupus.

34. The commercial package according to claim 33, wherein said lupus is selected from the group consisting of systemic lupus erythematosus, discoid lupus erythematosus, subacute cutaneous lupus erythematosus, drug-induced lupus and neonatal lupus.

35. The commercial package according to claim 30, wherein said agent is adapted for administration to an immune cell.

36. The commercial package according to claim 35, wherein said immune cell is 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.

37. The commercial package according to claim 36, wherein said immune cell is a lymphocyte.

38. The commercial package according to claim 37, wherein said lymphocyte is selected from the group consisting of a T lymphocyte and a B lymphocyte.

39. An isolated nucleic acid comprising the sequence of SEQ ID NO: 29.

40. A pharmaceutical composition for use in the treatment or the prevention of an autoimmune disease, said composition comprising:

(i) an agent capable of inhibiting the expression of a gene located in a PARl region and/or the activity of a protein encoded by said gene; and

(ii) a pharmaceutically acceptable carrier.

41. The composition according to claim 40, wherein said gene is selected from the group consisting of CD99, IL3RA, SLC25A6, AK123701, FLJ13330, ASMTL, P2RY8, DXYS155E, ASMT, ZBEDl, BC019893 and AK096998.

42. The composition according to claim 40, said composition being adapted for administration to an immune cell.

43. The composition according to claim 42, wherein said immune cell is 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.

44. The composition according to claim 43, wherein said immune cell is a lymphocyte.

45. The composition according to claim 44, wherein said lymphocyte is selected from the group consisting of a T lymphocyte and a B lymphocyte.

46. Method of diagnosing or prognosticating an autoimmune disease in a subject, said method comprising assessing whether a test parameter is increased with respect to a control parameter, said test parameter being selected from the group consisting of: (i) a copy number of a gene located in a PARl region;

(ii) a level of expression of a gene located in a PARl region;

(iii) a level of expression a protein encoded by a gene located in a PARl region; and (iv) an activity of a protein encoded by a gene located in a PARl region; wherein said increase is an indication that said subject has an increased likelihood of developing said autoimmune disease.

47. The method according to claim 46, wherein said gene is selected from the group consisting of CD99, IL3RA, SLC25A6, AK123701, FLJ13330,

ASMTL, P2RY8, DXYS155E, ASMT, ZBEDl, BC019893 and AK096998.

48. The method according to claim 46, wherein said test parameter is measured from a biological sample obtained from said subject.

49. The method according to claim 48, wherein said biological sample is a blood sample.

50. The method according to claim 49, wherein said blood sample is a myeloid lymphocyte layer.

51. The method according to claim 46, wherein said 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 connective tissue disease.

52. The method according to claim 51, wherein said autoimmune disease is lupus.

53. The method according to claim 52, wherein said lupus is selected from the group consisting of systemic lupus erythematosus, discoid lupus erythematosus, subacute cutaneous lupus erythematosus, drug-induced lupus and neonatal lupus.

54. The method according to claim 46, wherein said control parameter is selected from the group consisting of:

(i) two copies of a gene located in a PARl region;

(ii) a level of expression of a gene located in a PARl region in a control subject;

(iii) a level of expression of a protein encoded by a gene located in a PARl region in a control subject; and

(iv) an activity of a protein encoded by a gene located in a PARl region in a control subject.

55. The method according to claim 54, wherein said control subject is a subject at an earlier time or a subject substantially free of said autoimmune disease.

56. The method according to claim 54, wherein said gene is selected from the group consisting of CD99, IL3RA, SLC25A6, AK123701, FLJ13330, ASMTL, P2RY8, DXYS155E, ASMT, ZBEDl, BC019893 and AK096998.

57. The method according to claim 46, said method comprising performing a polymerase chain reaction.

58. The method according to claim 57, wherein said test parameter and said control parameter is the copy number of said gene.

59. The method according to claim 58, wherein said polymerase chain reaction is a real-time quantitative polymerase chain reaction.

60. The method according to claim 57, wherein said test parameter and said control parameter is the level of expression of said gene.

61. The method according to claim 60, wherein said polymerase chain reaction is a reverse-transcriptase polymerase chain reaction.

62. 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 said gene; and

• instructions for use of said means in the diagnosis or prognostication of an autoimmune disease in a subject.

63. The commercial package according to claim 62, wherein said means is a set of primers selected from the group consisting of: (i) 5'-CCTCTGTGGGCGTCCGTTCAT-S' (SEQ ID NO: 54) and 5'- CGGGTTCTGCTTGTCTCCATA-3' (SEQ ID NO: 55);

(v) 5'-AATAGTGGCCGAGGAGGGTGAC-S' (SEQ ID NO: 62) and 5'- ACCTGGCAAGTGCTCAGAGTTC-3' (SEQ ID NO: 63); (vi) 5'-AGGAGGTTCTATTTCGGGTGTC-S' (SEQ ID NO: 64) and 5'- CTTGGGCTGGTGGCAGAGGA-3' (SEQ ID NO: 65);

(vii) 5'-TACTGCTCCCATCGCTGGTGTC-S' (SEQ ID NO: 66) and 5'- GCCTCATTTCACTTTCACATCC-3' (SEQ ID NO: 67);

(viii) 5'-ACTCTCCAGCGGTTCTCAAAG-S' (SEQ ID NO: 68) and 5'GCTAAAGCCGTGGTATCACAG3' (SEQ ID NO: 69);

(x) 5'-AGAGCAGCCGTCTCCCATCCT-S' (SEQ ID NO: 72) and 5'- ACCCACCGCACTCACCTTCAT-3' (SEQ ID NO: 73); (xi) 5' -TGCCCACCTGCTCTGTAGAATC-S' (SEQ ID NO: 74) and 5'- TCATCACGCCCTGTCCCTCA-3' (SEQ ID NO: 75);

64. A method of screening for an agent capable of treating or preventing an autoimmune disease in a subject, said method comprising 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 said gene, said inhibition being indicative that the agent is capable of treating or preventing said autoimmune disease.

65. The method according to claim 64, wherein said gene is selected from the group consisting of CD99, IL3RA, SLC25A6, AK123701, FLJ13330, ASMTL, P2RY8, DXYS155E, ASMT, ZBEDl, BC019893 and AK096998.

66. The method according to claim 64, wherein said 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 connective tissue disease.

67. The method according to claim 66, wherein said autoimmune disease is lupus.

68. The method according to claim 67, wherein said lupus is selected from the group consisting of systemic lupus erythematosus, discoid lupus erythematosus, subacute cutaneous lupus erythematosus, drug-induced lupus and neonatal lupus.

69. The method according to claim 64, wherein said assessment is performed in an immune cell.

70. The method according to claim 69, wherein said immune cell is 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.

71. The method according to claim 70, wherein said immune cell is a lymphocyte.

72. The method according to claim 71, wherein said lymphocyte is selected from the group consisting of a T lymphocyte and a B lymphocyte.

73. The method according to claim 64, said method comprising performing a polymerase chain reaction.

74. The method according to claim 73, wherein said polymerase chain reaction is a reverse-transcriptase polymerase chain reaction.

75. A commercial package comprising:

• instructions for use of said means in screening for an agent capable of treating or preventing an autoimmune disease.

76. The commercial package according to claim 75, wherein said gene is selected from the group consisting of CD99, IL3RA, SLC25A6, AK123701, FLJ13330, ASMTL, P2RY8, DXYS155E, ASMT, ZBEDl, BC019893 and AK096998.

77. The commercial package according to claim 75, wherein said 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 connective tissue disease.

78. The commercial package according to claim 77, wherein said autoimmune disease is lupus.

79. The commercial package according to claim 78, wherein said lupus is selected from the group consisting of systemic lupus erythematosus, discoid lupus erythematosus, subacute cutaneous lupus erythematosus, drug-induced lupus and neonatal lupus.

80. The commercial package according to claim 75, wherein said instructions specify the use of an immune cell.

81. The commercial package according to claim 80, wherein said immune cell is 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.

82. The commercial package according to claim 81, wherein said immune cell is a lymphocyte.

83. The commercial package according to claim 82, wherein said lymphocyte is selected from the group consisting of a T lymphocyte and a B lymphocyte.

84. The commercial package according to claim 75, wherein said instructions specify the use of a polymerase chain reaction.

85. The commercial package according to claim 84, wherein said polymerase chain reaction is a reverse-transcriptase polymerase chain reaction.