WO2005049860A2 - Methodes et analyses diagnostiques et therapeutiques - Google Patents

Methodes et analyses diagnostiques et therapeutiques Download PDF

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WO2005049860A2
WO2005049860A2 PCT/EP2004/011961 EP2004011961W WO2005049860A2 WO 2005049860 A2 WO2005049860 A2 WO 2005049860A2 EP 2004011961 W EP2004011961 W EP 2004011961W WO 2005049860 A2 WO2005049860 A2 WO 2005049860A2
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ciita
disorder
sequence
nucleotide
nucleic acid
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WO2005049860A3 (fr
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Tomas Olsson
Fredrik Piehl
Olle Lidman
Maria Jansson
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Karolinska Innovations Ab
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • the present invention is based on the work described herein in relation to the gene mhc2ta or CIITA. It relates inter alia to association of disease susceptibility with alleles of the gene, methods of determining this in individuals, assays for therapeutics, and treatments. It especially relates to inflammatory and autoimmune disorders, acute myocardial infarction, rheumatoid arthritis and multiple sclerosis.
  • MS multiple sclerosis
  • AD Alzheimer dementia
  • MS a systemic and/or local inflammatory reactions occur, at least to some degree, in virtually all neurological diseases characterized by neuronal damage and death. These include mechanical trauma, multiple sclerosis (MS) , stroke and neurodegenerative disorders such as Alzheimer dementia (AD). In some of these conditions, e.g. MS, inflammation is believed to play a pivotal role for pathogenesis, whereas it has been ascribed an auxiliary role in the remaining.
  • Naturally occurring polymorphisms in genes that are critical for the regulation of inflammatory reactions in the nervous system may have impact on susceptibility or severity in a number of debilitating neurological diseases.
  • the central nervous system has been regarded as an immuno- priviliged site where MHC class II and T cells are largely absent under basal conditions (Shrikant and Benveniste, 1996) .
  • MHC class II expression and a variable extent of T cell infiltration occurs in nervous tissue in a broad variety of CNS diseases, including multiple sclerosis (MS) (Hof an et al., 1986) and viral encephalitides, but also in primarily non-inflammatory conditions like amyotrophic lateral sclerosis and Alzheimer (Dickson et al . , 1993; McGeer et al., 1993).
  • MHC class II in vivo is almost exclusively located to activated microglia and this observation correlates with findings supporting that this cell type functionally can act as APC, at least with regard to the reactivation of T cells primed in the periphery (Cash and Rott, 1994; Williams et al . , 1994; Ford et al . , 1996;shrikant and Benveniste, 1996; Aloisi et al . , 1998).
  • MHC class II expression pattern on microglia varies among rat strains both under normal conditions (Sedgwick et al., 1993) and non-specific damage (Piehl et al., 1999), and the degree of MHC class II up regulation after nerve-injury correlates to susceptibility to neuroinflammatory disease in a panel of inbred rat strains (Lundberg et al . , 2001) . Hypothetically, genetically determined differences in the expression levels of MHC class II may therefore dictate susceptibility to inflammation. Expression of MHC class II on microglia can be elicited by a number of inflammatory stimuli that converge on the IFN- ⁇ signaling pathway.
  • IL-l ⁇ , IL-4, IL-10, TGF- ⁇ and glucocorticoids can modulate the expression (Ting and Trowsdale, 2002; Boss and Jensen, 2003) .
  • the promoter elements of the class II genes are highly conserved between 5 species and contain the W/S, XI, X2 and Y boxes.
  • the transcription factor NF-Y/CBF binds to the Y box, whereas cyclic AMP response element binding protein (CREB) binds to the X2 box.
  • CREB cyclic AMP response element binding protein
  • the XI element interacts with a trimer, the RFX factor.
  • One of the components of RFX, RFX5 also binds to
  • class II transactivator (CIITA) is also a necessary component of the transcriptional machinery needed to initiate class II transcription, since it acts as a platform for the assembly of the factors mentioned above. Genetic heterogeneity in any
  • the inventors have established a role for CITTA in inflammatory and autoimmune disorders, and association of alleles of the gene with susceptibility to different disorders, in particular acute myocardial infarction (AMI), rheumatoid arthritis (RA) and multiple sclerosis (MS) .
  • AMI acute myocardial infarction
  • RA rheumatoid arthritis
  • MS multiple sclerosis
  • the invention in various aspects and embodiments relates to methods comprising determining the presence of allelic forms of the MHC class II transactivator (CIITA) in a subject, for diagnostic and predictive purposes, also to methods of down- regulating CIITA gene function for therapeutic purposes.
  • Diseases and conditions for which such methods may be employed include AMI, RA, and MS.
  • CIITA gene sequences are available for the following species under the indicated accession numbers: Rat: NM_053529, Mouse: NM_007575, Human: AF410154.
  • accession numbers provide CIITA sequences:
  • MHC2TA Homo sapiens MHC class II transactivator
  • MHC2TA Homo sapiens MHC2TA
  • C2TA Homo sapiens MHC class II transactivator type II
  • C2TA Homo sapiens MHC class II transactivator type IV
  • C2TA Homo sapiens MHC class II transactivator type III
  • C2TA Homo sapiens MHC class II transactivator type I
  • CIITA The chromosomal location of CIITA in different species is rat: lOqll, mouse: 16B1, human: 16pl3.
  • the bare lymphocyte syndrome is a hereditary disorder in which the patients complete lack the expression of MHC class II.
  • CIITA was identified (Steimle et al., 1993). Transfer of functional CIITA to MHC negative mutant cell line restores the MHC class II expression. Furthermore, the transactivating role of CIITA was described and the loss of CIITA-function in a subgroup of patients with bare lymphocyte syndrome is explained to be due to a 24 amino acid deletion in the gene (Steimle et al . , 1993) .
  • CIITA is upregulated by: IL-1, IFN ⁇ , LPS, and IL-4.
  • IFN ⁇ induces interferon regulatory factor (IRF) 1/2 and signal transducer and activator of transcription (STAT) 1 via Janus associated kinase (Jak) 1/2.
  • IRF interferon regulatory factor
  • STAT1 signal transducer and activator of transcription
  • GAS Janus associated kinase
  • USF constitutiveiy expressed upstream stimulatory factor
  • IFN ⁇ is downregulated by: IFN ⁇ , IL-10, NO, TGF ⁇ .
  • IFN ⁇ is proposed to act at a post-transcriptional level.
  • IFN stimulated gene factor 3 (ISGF3) gamma is essential for IFN- ⁇ to mediate inhibition of MHC class II induction (Lu et al . , 1995) .
  • TGF ⁇ down regulate both constitutive and IFN ⁇ induced CIITA expression through Smad3 (Dong et al . , 2001)
  • the MHC class II transactivator is the master regulator of MHC class II expression, e.g. CIITA-/- mice have low levels MHC class II expression and low numbers of CD4+ T cells (Chang et al . , 1996), (Itoh-Lindstrom et al . , 1999).
  • CIITA does not bind DNA directly but assembles transcription factors at promoters containing the cis-acting elements X, Y, and W/Z/S. These promoter elements are present in all classical and non-classical MHC class II promoters including the Ii promoter.
  • CIITA acts by recruiting components of the transcription machinery and the histone acetyltransferases (HATs) .
  • CIITA has also intrinsic HAT activity and is able to titrate CBP (creb binding protein) .
  • CBP creb binding protein
  • CIITA binds to: • Transcription factors o Nuclear Factor (NF) Y, that binds the Y promoter element (CCAAT box) o RFX factor that interacts with the XI promoter element and with NF-Y o CREB (cAMP-response element-binding protein) , interacts also with RFX
  • pathogens e.g. cytomegalovirus, Mycobacterium bovis, Chlamydia, varicella-zoster virus, and Epstein Barr virus, and HIV
  • pathogens e.g. cytomegalovirus, Mycobacterium bovis, Chlamydia, varicella-zoster virus, and Epstein Barr virus, and HIV
  • CIITA Reviewed in (Harton and Ting, 2000)
  • Toxoplasma gondii down-regulates the transcript levels CIITA in glial cell cultures (Luder et al . , 2003) .
  • CIITA can contribute to Th cell differentiation by suppressing IL-4 production in Thl cells (Gourley et al . , 1999; Sisk et al . , 2000). However, CIITA is expressed in low levels in activated mouse T cells and CIITA is not regulated differentially in human or murine Thl and Th2 cells (Otten et al., 2003) .
  • MHC class II expression Several neurological diseases are associated with increased MHC class II expression, including MS, AD, Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS) (Neumann et al., 2002) .
  • MS MS
  • AD Parkinson's disease
  • ALS amyotrophic lateral sclerosis
  • Blockade of neuronal activity promotes IFN- ⁇ induced MHC class II on microglia and astrocytes in organotypic hippocampal slice cultures (Neumann et al . , 1996). Furthermore, neurotrophins inhibit MHC class II expression on microglia (Neumann et al., 1998). There are data that argue for a site specific immune regulation in the brain (Phillips et al., 1999). The brainstem displays a more enhanced MHC class II induction after IFN- ⁇ treatment in contrast to hippocampus subjected to the same treatment.
  • MHCII and CIITA in astroglial and microglial cells in vitro have been, for example, studied by Benveniste and co-workers (e.g. references (O'Keefe et al., 2001) and (Dong et al . , 1999) ) .
  • CIITA knockout mice and mice transgenic which express CIITA in astrocytes under the control of the GFAP promoter have been examined in a MOG 35-55 induction protocol.
  • CIITA deficient animals were resistant to EAE induction but T cells from these animals proliferated and secreted Thl cytokines in response to stimulation with MOG.
  • CIITA induced MHC class II expression in astrocytes did not increase the EAE susceptibility (Stuve et al., 2002).
  • C57BL/6 mice deficient for the class II transactivator, invariant chain (Ii) , and H-2M (DM) are resistant to EAE induction.
  • HMG-CoA reductase inhibitor (Atorvastatin) promotes a Th2 bias and reverses paralysis in chronic and relapsing EAE in mice. Atorvastatin induce STAT6 phosphorylation and secretion of IL-4, IL-5, IL-10, and TGF- ⁇ . STAT-4 phosphorylation was inhibited and production of IL- 2, IL-12, IFN- ⁇ , and TNF- ⁇ was suppressed. Furthermore, statin treatment reduces CNS MHC class II expression and inhibits IFN- ⁇ induced CIITA expression in microglial cell cultures (Youssef et al., 2002).
  • Phagocytic macrophages in active demyelinating MS lesion displayed a moderate to strong immunostaing of CIITA and RFX, co-localized with general transcription factors such as NF-kb, IRF-1, STAT-1, USF, and CREB.
  • a genome screen for linkage analysis using 397 microsatellite markers was performed in 47 Australian sibling-pairs with MS (Ban et al . , 2002). Four regions displayed suggestive linkage and 18 regions for potential linkage. 16pl3 (LOD 1.5, max marker D163103) was included among the 18.
  • MHCIITA The coding and promoter regions of MHCIITA were sequenced for polymorphisms in 50 normal Caucasian individuals. Four SNPs were identified in the coding region of CIITA and one SNP in pill, but none in pi or pIV. (Patarroyo et al., 2002)
  • Pancreatic lymphocytic infiltration occur in NOD CIITA deficient mice, however progression to diabetes is abrogated (Mora et al . , 1999) .
  • 16pl3 was identified as a susceptible locus for coronary heart disease and high blood pressure (Francke et al., 2001)
  • the experimental demonstration of association between CIITA alleles and particular diseases paves the way for aspects of the present invention to provide the use of materials and methods, such as are disclosed and discussed herein, for establishing the presence or absence in a test sample of a particular form of the gene, in particular an allele or variant specifically associated with AMI or other disease. This may be for diagnosing a predisposition of an individual to AMI or other disease. It may be for diagnosing of a patient with the disease as being associated with CIITA.
  • the present invention relates to the identification of functional polymorphisms in the form of allelic variations e.g. Single Nucleotide Polymorphisms (SNPs) in the MHC2TA or CIITA gene and an association between allelic variations e.g. SNPs in the MHC2TA or CIITA gene where certain alleles and SNPs confer predisposition, susceptibility or resistance to disease.
  • allelic variations e.g. Single Nucleotide Polymorphisms (SNPs) in the MHC2TA or CIITA gene
  • SNPs Single Nucleotide Polymorphisms
  • a gene or locus which confers susceptibility to a disease condition may contain one or more sites at which polymorphisms exist. The presence of these polymorphisms leads to different alleles of the gene or locus, one of which may be associated with an increased susceptibility to a disease condition.
  • An individual who is susceptible to a disease condition may have a predisposition to that condition which places that individual at a higher risk of incurring the condition during their lifetime than the population as a whole. Although at a higher risk of doing so, a susceptible individual may, in fact, never incur the disease condition.
  • an allele may confer protection from disease.
  • a disease gene with two alleles it can be difficult to distinguish between the gene having a susceptibility allele or a protective allele, where the other allele is neutrally or positively associated with disease.
  • a gene associated with a disease may contain a number of SNPs within either its coding or non- coding region.
  • Disease association may be caused by a particular SNP or by a particular haplotype consisting of a number of SNPs .
  • An SNP in coding sequence may alter the sequence of a polypeptide, giving rise to a defective or variant isoform which may be associated with a disease condition.
  • An SNP in non-coding sequence may also lead to a disease condition, for example, by altering the activity of an enhancer element which directs polypeptide expression.
  • an SNP may have 5 no phenotypic effect.
  • association analysis of particular SNPs and haplotypes using populations of affected and non-affected individuals may indicate that an SNP or haplotype is associated with a disease 10 condition.
  • the present inventors have shown that particular alleles are associated with disease conditions. This allows the development of methods associated with the diagnosis and 15 therapy of CIITA related conditions.
  • a first aspect of the present invention provides a method for determining the susceptibility of an individual to a disease or disorder comprising:
  • nucleotide 5' to the A of the ATG of the CIITA III initiation codon is designated -1 and the sequence of the CIITA locus has the database accession number AF000003 or NM000246. Particularly, position -155 of the CIITA pill locus, wherein the nucleotide 5' to the A of the ATG of
  • the CIITA III initiation codon is designated -1 and the sequence of the CIITA locus has the database accession number AF000003.
  • SNPs are shown in Table 1 35
  • the susceptibility of the individual to a disorder such as RA, MS and ischemic heart disease is indicated by the identity of the nucleotide present at said one or more positions.
  • the nucleotide at the one or more positions of polymorphism may be an allele which is shown in Table 1.
  • methods may comprise determining the identity of the nucleotide at a position of single nucleotide polymorphism at e.g. position -155 of the CIITA III promoter, for example, the presence or absence of the nucleotide G at this position.
  • identity of nucleotides at other positions of single nucleotide polymorphism described herein and shown in Table 1 may also be determined.
  • Particular alleles of polymorphisms which are located in the CIITA regulatory regions may alter expression from the gene or affect the processing or stability of the mRNA transcript.
  • the presence of such alleles may be determined by measuring the amount and/or stability of the CIITA mRNA.
  • a variant form of the gene may contain one or more insertions, deletions, substitutions and/or additions of one or more nucleotides compared with another form of the sequence (such as shown in Table 1) which may or may not disrupt or otherwise alter the gene function. Differences at the nucleic acid level are not necessarily reflected by a difference in the amino acid sequence of the encoded polypeptide. However, a mutation or other difference in a gene may result in a frame-shift or stop codon, which could seriously affect ' the nature of the polypeptide produced (if any) , or a point mutation or gross mutational change to the encoded polypeptide, including insertion, deletion, substitution and/or addition of one or more amino acids or regions in the polypeptide.
  • a mutation in a promoter sequence or other regulatory region may prevent or reduce expression from the gene or affect the processing or stability of the mRNA transcript.
  • a sequence alteration may affect alternative splicing of mRNA.
  • allelic variations resulting from alternative splicing are provided by the present invention.
  • sequence information can be retained and subsequently searched without recourse to the original nucleic acid itself.
  • sequence alteration or mutation may be identified by scanning a database of sequence information using a computer or other electronic means.
  • tests may be carried out on preparations containing genomic DNA, cDNA and/or mRNA.
  • Testing cDNA or mRNA has the advantage of the complexity of the nucleic acid being reduced by the absence of intron sequences, but the possible disadvantage of extra time and effort being required in making the preparations. RNA is more difficult to manipulate than DNA because of the wide-spread occurrence of RN'ases.
  • Methods according to some aspects of the present invention may comprise determining the binding of a oligonucleotide probe to the genomic sample.
  • the probe may comprise a nucleotide sequence which binds specifically to a particular allele of the at least one polymorphism and does not bind specifically to other alleles of the at least one polymorphism.
  • the oligonucleotide probe may comprise a label and binding of the probe may be determined by detecting the presence of the label.
  • a method may include hybridisation of one or more (e.g. two) oligonucleotide probes or primers to target nucleic acid. Where the nucleic acid is double-stranded DNA, hybridisation will generally be preceded by denaturation to produce single- stranded DNA.
  • the hybridisation may be as part of a PCR procedure, or as part of a probing procedure not involving PCR.
  • An example procedure would be a combination of PCR and low stringency hybridisation.
  • a screening procedure chosen from the many available to those skilled in the art, is used to identify successful hybridisation events and isolated hybridised nucleic acid.
  • Binding of a probe to target nucleic acid may be measured using any of a variety of techniques at the disposal of those skilled in the art.
  • probes may be radioactively, fluorescently or enzymatically labelled.
  • Other methods not employing labelling of probe include examination of restriction fragment length polymorphisms, amplification using PCR, RN'ase cleavage and allele specific oligonucleotide probing.
  • Probing may employ the standard Southern blotting technique. For instance DNA may be extracted from cells and digested with different restriction enzymes. Restriction fragments may then be separated by electrophoresis on an agarose gel, before denaturation and transfer to a nitrocellulose filter. Labelled probe may be hybridised to the DNA fragments on the filter and binding determined.
  • DNA for probing may be prepared from RNA preparations from cells.
  • Suitable selective hybridisation conditions for oligonucleotides of 17 to 30 bases include hybridization overnight at 42°C in 6X SSC and washing in 6X SSC at a series of increasing temperatures from 42°C to 65°C.
  • An oligonucleotide for use in nucleic acid amplification may be about 30 or fewer nucleotides in length (e.g. 18, 21 or 24). Generally specific primers are upwards of 14 nucleotides in length, but need not be longer than 18-20. Those skilled in the art are well versed in the design of primers for use processes such as PCR. Various techniques for synthesizing oligonucleotide primers are well known in the art, including phosphotriester and phosphodiester synthesis methods.
  • Nucleic acid may also be screened using a variant- or allele- specific probe.
  • a probe may correspond in sequence to a region of the mhc2ta or CIITA gene, or its complement, which contains one or more of the single nucleotide polymorphisms described herein, which are shown to be associated with disease susceptibility.
  • specific hybridisation of such a probe to test nucleic acid is indicative of the presence of the sequence alteration in the test nucleic acid.
  • more than one probe may be used on the same test sample.
  • Nucleic acid in a test sample which may be a genomic sample or an amplified region thereof, may be sequenced to identify or determine the identity of a polymorphic allele.
  • An allele may be identified by comparing sequences of material obtained from different individuals or populations.
  • the allele of the SNP in the test nucleic acid can therefore be compared with a susceptibility allele to determine whether the test nucleic acid contains one or more alleles which are associated with disease.
  • nucleic acid for testing may be prepared from nucleic acid removed from cells or in a library using a variety of other techniques such as restriction enzyme digest and electrophoresis .
  • Sequencing of an amplified product may involve precipitation with isopropanol, resuspension and sequencing using a TaqFS+ Dye terminator sequencing kit. Extension products may be electrophoresed on an ABI 377 DNA sequencer and data analysed using Sequence Navigator software.
  • Nucleic acid in a test sample may be probed under conditions for selective hybridisation and/or subjected to a specific nucleic acid amplification reaction such as the polymerase chain reaction (PCR) (reviewed for instance in "PCR protocols; A Guide to Methods and Applications", Eds. Innis et al, 1990, Academic Press, New York, Mullis et al, Cold Spring Harbor Symp. Quant. Biol., 51:263, (1987), Ehrlich (ed) , PCR technology, Stockton Press, NY, 1989, and Ehrlich et al, Science, 252:1643-1650, (1991)).
  • PCR comprises steps of denaturation of template nucleic acid (if double-stranded) , annealing of primer to target, and polymerisation.
  • the nucleic acid probed or used as template in the amplification reaction may be genomic DNA, cDNA or RNA.
  • nucleic acid amplification techniques include strand displacement activation, the QB replicase system, the repair chain reaction, the ligase chain reaction, rolling circle amplification and ligation activated transcription.
  • PCR is used herein in contexts where other nucleic acid amplification techniques may be applied by those skilled in the art. Unless the context requires otherwise, reference to PCR should be taken to cover use of any suitable nucleic amplification reaction available in the art.
  • Methods of the present invention may therefore comprise amplifying the portion of the CIITA gene and region in said genomic sample containing the one or more positions of allelic variation, e.g. single nucleotide polymorphism.
  • Allele- or variant-specific oligonucleotides may be used in PCR to specifically amplify particular sequences if present in a test sample. Assessment of whether a PCR band contains a gene variant may be carried out in a number of ways familiar to those skilled in the art.
  • the PCR product may for instance be treated in a way that enables one to display the polymorphism on a denaturing polyacrylamide DNA sequencing gel, with specific bands that are linked to the gene variants being selected.
  • the region of genomic sample comprising a polymorphism may be amplified using a pair of oligonucleotide primers, of which the first member of the pair comprises a nucleotide sequence which hybridises to a complementary sequence which is proximal to and 5' of the position of single nucleotide polymorphism, and the second member of the primer pair comprises a nucleotide sequence which hybridises to a complementary sequence which is proximal to and 3' of the position of single nucleotide polymorphism.
  • the first member of the pair of oligonucleotide primers may comprise a nucleotide sequence which hybridises to a complementary sequence which is proximal to and 5' or 3' of the polymorphism
  • the second member of the pair may comprise a nucleotide sequence which hybridises under stringent conditions to a particular allele of the polymorphism and not to other alleles, such that amplification only occurs in the presence of the particular allele.
  • a further aspect of the present invention provides a pair of oligonucleotide amplification primers suitable for use in the methods described herein.
  • a suitable pair of amplification primers according to this aspect may have a first member comprising a nucleotide sequence which hybridises to a complementary sequence which is proximal to and 5' of a (e.g. single nucleotide) the polymorphism in the CIITA III promoter at position -155 for example as shown in Table 1, and a second member comprising a nucleotide sequence which hybridises to a complementary sequence which is proximal to and 3' of the polymorphism.
  • a first member comprising a nucleotide sequence which hybridises to a complementary sequence which is proximal to and 5' of a (e.g. single nucleotide) the polymorphism in the CIITA III promoter at position -155 for example as shown in Table 1
  • a second member comprising a nucleotide sequence which hybridises to a complementary sequence which is proximal to and 3' of the polymorphism.
  • the allele of the at least one polymorphism (i.e. the identity of the nucleotide at the position of polymorphism) may then be determined by determining the binding of an oligonucleotide probe to the amplified region of the genomic sample.
  • a suitable oligonucleotide probe comprises a nucleotide sequence which binds specifically to a particular allele of the at least one polymorphism and does not bind specifically to other alleles of the at least one polymorphism.
  • Suitable pairs of amplification primers may have a first member comprising a nucleotide sequence which hybridises to a complementary sequence which is proximal to and 5' or 3' of a (e.g. single nucleotide) polymorphism at position -155 of the CIITA III promoter, or other position for example as shown in Table 1, and a second member of the pair comprising a nucleotide sequence which hybridises under stringent conditions to a particular allele of the polymorphism and not to other alleles, such that amplification only occurs in the presence of the particular allele.
  • a first member comprising a nucleotide sequence which hybridises to a complementary sequence which is proximal to and 5' or 3' of a (e.g. single nucleotide) polymorphism at position -155 of the CIITA III promoter, or other position for example as shown in Table 1
  • a second member of the pair comprising a nucleotide sequence which hybrid
  • An alternative or supplement to looking for the presence of variant sequences in a test sample is to look for the presence of the normal sequence, e.g. using a suitably specific oligonucleotide probe or primer.
  • oligonucleotide probes and primers have been discussed in more detail above.
  • a further aspect of the present invention provides an oligonucleotide which hybridises specifically to a nucleic acid sequence which comprises an allele of a polymorphism selected from the group consisting of the polymorphisms shown in Table 1, e.g. -155 of the CIITA III promoter, for example as shown in Table 1.
  • oligonucleotides may be used in a method of screening nucleic acid.
  • Some preferred oligonucleotides have a sequence which is complementary to the SNP's reference sequence shown in Table 1, or a sequence which differs from such a sequence by addition, substitution, insertion or deletion of one or more nucleotides, but preferably without abolition of ability to hybridise selectively to an allele of a polymorphism as described herein, that is wherein the degree of similarity of the oligonucleotide or polynucleotide with one of the sequences given is sufficiently high.
  • oligonucleotides according to the present invention are at least about 10 nucleotides in length, more preferably at least about 15 nucleotides in length, more preferably at least about 20 nucleotides in length. Oligonucleotides may be up to about 100 nucleotides in length, more preferably up to about 50 nucleotides in length, more preferably up to about 30 nucleotides in length.
  • the boundary value "about X nucleotides" as used above includes the boundary value "X nucleotides”.
  • Allele- or variant-specific oligonucleotide probes or primers according to embodiments of the present invention may be selected from those shown in Table 1.
  • Approaches which rely on hybridisation between a probe and test nucleic acid and subsequent detection of a mismatch may be employed. Under appropriate conditions (temperature, pH etc.), an oligonucleotide probe will hybridise with a sequence which is not entirely complementary. The degree of base-pairing between the two molecules will be sufficient for them to anneal despite a mis-match.
  • Various approaches are well known in the art for detecting the presence of a mis-match between two annealing nucleic acid molecules.
  • RN'ase A cleaves at the site of a mis-match. Cleavage can be detected by electrophoresing test nucleic acid to which the relevant probe or probe has annealed and looking for smaller molecules (i.e. molecules with higher electrophoretic mobility) than the full length probe/test hybrid.
  • an oligonucleotide probe that has the sequence of a region of the CIITA gene (either sense or anti-sense strand) in which the SNPs associated disease susceptibility as described herein are known to occur may be annealed to test nucleic acid and the presence or absence of a mis-match determined. Detection of the presence of a mis-match may indicate the presence in the test nucleic acid of a mutation associated with disease susceptibility.
  • an oligonucleotide probe that has the sequence of a region of the gene including a polymorphism associated with disease susceptibility may be annealed to test nucleic acid and the presence or absence of a mis-match determined.
  • the presence of a mis-match may indicate that the nucleic acid in the test sample has the normal sequence (the absence of a mis-match indicating that the test nucleic acid has the mutation) . In either case, a battery of probes to different regions of the gene may be employed.
  • Nucleic acid according to the present invention may be provided as part of a kit, e.g. in a suitable container such as a vial in which the contents are protected from the external environment.
  • the kit may include instructions for use of the nucleic acid, e.g. in PCR and/or a method for determining the presence of nucleic acid of interest in a test sample.
  • a kit wherein the nucleic acid is intended for use in PCR may include one or more other reagents required for the reaction, such as polymerase, nucleosides, buffer solution etc.
  • the nucleic acid may be labelled.
  • a kit for use in determining the presence or absence of nucleic acid of interest may include one or more articles and/or reagents for performance of the method, such as means for providing the test sample itself, e.g. a swab for removing cells from the buccal cavity or a syringe for removing a blood sample (such components generally being sterile) .
  • Another aspect of the present invention provides a method for determining the presence or absence of an allele of a polymorphic nucleic acid sequence in a test sample comprising: contacting a polymorphic nucleic acid sequence with a probe which specifically binds to the allele of the polymorphic nucleic acid sequence; and, determining binding of the nucleic acid sequence and the probe, said method being characterised in that the polymorphic nucleic acid sequence comprises one or more positions of polymorphism for example selected from the group consisting of nt -155, 1498, 2420, and 2675 wherein the nucleotide 5' to the
  • a of the ATG of the CIITA III initiation codon is designated -
  • nucleotide 5' to the A of the ATG of the CIITA III initiation codon is designated -1 and the sequence of the CIITA locus has the database accession number AF000003, the identity of the nucleotide at the one or more positions of single nucleotide polymorphism determining the allele of the polymorphic nucleic acid sequence.
  • Another aspect of the present invention provides a method for determining the presence or absence in a test sample of an allele of a polymorphic nucleic acid sequence comprising one or more positions of polymorphism, the method comprising: determining the identity of the nucleotide at one or more positions of polymorphism, for example selected from the group consisting of -155, 1498, 2420, and 2675 wherein the nucleotide 5' to the A of the ATG of the CIITA III initiation codon is designated -1 and the sequence of the CIITA locus has the database accession number AF000003 or NM000246 (Table 1) .
  • Particularly position -155 of the CIITA III locus wherein the nucleotide 5' to the A of the ATG of the CIITA III initiation codon is designated -1 and the sequence of the CIITA locus has the database accession number AF000003, the presence of the allele of the polymorphic nucleic acid sequence being determined by the identity of the nucleotide at the one or more positions of polymorphism.
  • such a method may comprise amplifying the polymorphic nucleic acid sequence using a pair of oligonucleotide primers.
  • physical detection may be employed using for example hybridisation of a suitable probe, or a transcription factor or other agent that binds nucleic acid in a sequence-specific fashion, or detection may be performed in silico or using suitable data analysis techniques, e.g. on a computer.
  • the identity of the nucleotides at positions of single nucleotide polymorphism at e.g. nt -155, 1498, 2420, and 2675 wherein the nucleotide 5' to the A of the ATG of the CIITA III initiation codon is designated -1 and the sequence of the CIITA locus has the database accession number AF000003 or NM000246 (Table 1) may be determined using such a method, (in particular, the presence of the nucleotide G at position -155 of the CIITA pill locus, wherein the nucleotide 5' to the A of the ATG of the CIITA III initiation codon is designated -1 and the sequence of the CIITA locus has the database accession number AF000003) .
  • the presence of differences in sequence of nucleic acid molecules may be detected by means of restriction enzyme digestion, such as in a method of DNA fingerprinting where the restriction pattern produced when one or more restriction enzymes are used to cut a sample of nucleic acid is compared with the pattern obtained when a sample containing the normal gene shown or a variant or allele, e.g. as containing an alteration shown in Table 1 is digested with the same enzyme or enzymes.
  • promoter is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i.e. in the 3' direction on the sense strand of double-stranded DNA) .
  • operably linked means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter.
  • DNA operably linked to a promoter is "under transcriptional initiation regulation" of the promoter.
  • Promoter activity is used to refer to ability to initiate transcription.
  • the level of promoter activity is quantifiable for instance by assessment of the amount of mRNA produced by transcription from the promoter or by assessment of the amount of protein product produced by translation of mRNA produced by transcription from the promoter.
  • the amount of a specific mRNA present in an expression system may be determined for example using specific oligonucleotides which are able to hybridise with the mRNA and which are labelled or may be used in a specific amplification reaction such as the polymerase chain reaction.
  • Use of a reporter gene facilitates determination of promoter activity by reference to protein production.
  • a promoter region may be operably linked to a heterologous gene, e.g. a coding sequence.
  • a “heterologous” or “exogenous” gene is generally not a modified form of CIITA.
  • the gene may be transcribed into mRNA which may be translated into a peptide or polypeptide product which may be detected and preferably quantitated following expression.
  • a gene whose encoded product may be assayed following expression is termed a "reporter gene", i.e. a gene which "reports" on promoter activity.
  • the reporter gene preferably encodes an enzyme which catalyses a reaction which produces a detectable signal, preferably a visually detectable signal, such as a coloured product.
  • a detectable signal preferably a visually detectable signal, such as a coloured product.
  • Many examples are known, including ⁇ -galactosidase and luciferase.
  • ⁇ -galactosidase activity may be assayed by production of blue colour on substrate, the assay being by eye or by use of a spectro-photometer to measure absorbance. Fluorescence, for example that produced as a result of luciferase activity, may be quantitated using a spectrophotometer.
  • Radioactive assays may be used, for instance using chloramphenicol acetyltransferase, which may also be used in non-radioactive assays.
  • the presence and/or amount of gene product resulting from expression from the reporter gene may be determined using a molecule able to bind the product, such as an antibody or fragment thereof.
  • the binding molecule may be labelled directly or indirectly using any standard technique.
  • CIITA gene with an allelic sequence or haplotype or SNP identity that is associated with an inflammatory or autoimmune disorder as discussed herein.
  • a test sample of nucleic acid may be provided for example by extracting nucleic acid from cells or biological tissues or fluids, urine, saliva, faeces, a buccal swab, biopsy or preferably blood, or for pre-natal testing from the amnion, placenta or foetus itself.
  • determining the presence or absence in a test sample of a particular polypeptide such as the CIITA polypeptide or an amino acid sequence mutant, variant, isoform or allele thereof.
  • a sample may be tested for the presence of a binding partner for a specific binding member such as an antibody molecule (or mixture of antibodies) , specific for one or more particular variants of the CIITA polypeptide.
  • a sample may be tested for the presence of a binding partner for a specific binding member such as an antibody molecule (or mixture of antibodies), specific for the CIITA polypeptide.
  • the sample may be tested by being contacted with a specific binding member such as an antibody molecule under appropriate conditions for specific binding, before binding is determined, for instance using a reporter system as discussed. Where a panel of antibody molecules is used, different reporting labels may be employed for each antibody so that binding of each can be determined.
  • a specific binding member such as an antibody may be used to isolate and/or purify its binding partner polypeptide from a test sample, to allow for sequence and/or biochemical analysis of the polypeptide to determine whether it has the sequence and/or properties of the polypeptide whose sequence is disclosed herein, or if it is a mutant or variant form.
  • Amino acid sequence is routine in the art using automated sequencing machines .
  • An antibody molecule may be a whole antibody or an antibody fragment.
  • Example antibody fragments, capable of binding an antigen or other binding partner are the Fab fragment consisting of the VL, VH, Cl and CHI domains; the Fd fragment consisting of the VH and CHI domains; the Fv fragment consisting of the VL and VH domains of a single arm of an antibody; the dAb fragment which consists of a VH domain; isolated CDR regions and F(ab')2 fragments, a bivalent fragment including two Fab fragments linked by a disulphide bridge at the hinge region. Single chain Fv fragments are also included.
  • test sample containing one or more polypeptides may be provided for example as a crude or partially purified cell or cell lysate preparation, e.g. using tissues or cells, such as from saliva, faeces, or preferably blood, or for pre-natal testing from the amnion, placenta or foetus itself.
  • Establishing that an individual has a particular CIITA sequence or feature of sequence that is associated with an inflammatory or autoimmune disorder allows for targeted therapy, by way of anti-CIITA treatment. Further aspects and embodiments of the present invention are concerned with such treatment and with obtaining and using substances that target CIITA and are useful in therapy of disorders in accordance with the present invention.
  • the present invention provides a method of treating a disorder, particularly an inflammatory or autoimmune disorder, especially AMI, RA or MS, by means of modulating the activity of CIITA, e.g. targeting CIITA to reduce or increase its activity in the cell, or by targeting the CIITA protein interaction with a CIITA binding partner (e.g. as identified supra) , e.g. to inhibit or promote the interaction, and/or by modulating the level of CIITA protein within the cell, e.g. reducing the level such as by eliminating protein if produced and/or inhibiting its production.
  • Activity of CIITA may be modulated by targeting a product of another gene, e.g. a protein that affects CIITA expression, stability or activity.
  • Methods of treatment of the human or animal body by way of therapy may be excluded.
  • a method of the invention may be carried out in vivo, for example in a method of therapy (which may be prophylactic) or in a non-therapeutic method.
  • Other methods may be carried out in vitro or ex vivo .
  • CIITA is targeted for modulation, such as inhibition, that is to say its function is altered, and in different embodiments may be inhibited, for instance by inhibiting binding of CIITA protein to a CIITA binding partner, or enhanced.
  • a substance may inhibit binding by inhibiting physical interaction between CIITA and another molecule, or by binding in a way that has a steric effect on the conformation of binding site.
  • the activity or function of CIITA may be targeted, as noted, by means of a substance that interacts in some way with the protein.
  • Another approach employs regulation at the nucleic acid level to inhibit activity or function by down-regulating production of the component.
  • expression of a CIITA gene may be inhibited using anti-sense technology.
  • anti-sense genes or partial gene sequences to down-regulate gene expression is well-established.
  • Anti-sense oligonucleotides may be designed to hybridise to the complementary sequence of nucleic acid, pre-mRNA or mature mRNA, interfering with the production of CIITA so that its expression is reduced or completely or substantially completely prevented.
  • antisense techniques may be used to target control sequences of a gene, e.g. in the 5' flanking sequence, whereby the antisense oligonucleotides can interfere with expression control sequences.
  • the construction of antisense sequences and their use is described for example in Peyman and Ulman, Chemical Reviews, 90:543-584, (1990) and Crooke, Ann. Rev. Pharmacol. Toxicol., 32:329-376, (1992).
  • Oligonucleotides may be generated in vitro or ex vivo for administration or anti-sense RNA may be generated in vivo within cells in which down-regulation is desired.
  • double-stranded DNA may be placed under the control of a promoter in a "reverse orientation" such that transcription of the anti-sense strand of the DNA yields RNA which is complementary to normal mRNA transcribed from the sense strand of the target gene.
  • the complementary anti-sense RNA sequence is thought then to bind with mRNA to form a duplex, inhibiting translation of the endogenous mRNA from the target gene into protein. Whether or not this is the actual mode of action is still uncertain. However, it is established fact that the technique works .
  • the complete sequence corresponding to the coding sequence in reverse orientation need not be used. For example fragments of sufficient length may be used.
  • a suitable fragment may have about 14-23 nucleotides, e.g. about 15, 16 or 17.
  • An alternative to anti-sense is to use a copy of all or part of the target gene inserted in sense, that is the same, orientation as the target gene, to achieve reduction in expression of the target gene by co-suppression; Angell &
  • RNA interference is a two-step process.
  • dsRNA is cleaved within the cell to yield short interfering RNAs (siRNAs) of about 21-23nt length with 5' terminal phosphate and 3' short overhangs ( ⁇ 2nt) .
  • siRNAs target the corresponding mRNA sequence specifically for destruction (Zamore P.D. Nature Structural Biology, 8, 9, 746-750, (2001)
  • RNAi may also be efficiently induced using chemically synthesized siRNA duplexes of the same structure with 3'- overhang ends (Zamore PD et al Cell, 101, 25-33, (2000)).
  • Synthetic siRNA duplexes have been shown to specifically suppress expression of endogenous and heterologous genes in a wide range of mammalian cell lines (Elbashir SM. et al. Nature, 411, 494-498, (2001)). See also Fire (1999) Trends Genet . 15: 358-363, Sharp (2001) Genes Dev. 15: 485-490, Hammond et al. (2001) Na ture Rev. Genes 2: 1110-1119 and Tuschl (2001) Chem . Biochem . 2: 239- 245, Hannon (2002) Nature 418 (6894 ) :244-51, Ueda (2001) J Neurogenet . 15(3-4): 193-204, Lindenbach (2002) Mol Cell .
  • nucleic acid is used which on transcription produces a ribozyme, able to cut nucleic acid at a specific site - thus also useful in influencing gene expression.
  • Background references for ribozymes include Kashani-Sabet and Scanlon, 1995, Cancer Gene Therapy, 2(3): 213-223, and Mercola and Cohen, 1995, Cancer Gene Therapy, 2(1), 47-59.
  • CIITA activity may also be increased, when the preferred outcome is stimulation. This can be achieved by interfering with signalling molecules, or intracellular messengers of such molecules, that inhibit CIITA transcription using a similar methodological approach.
  • signalling molecules or intracellular messengers of such molecules, that inhibit CIITA transcription using a similar methodological approach.
  • substances with a negative regulatory effect on CIITA transcription are IFN- ⁇ , IL-10, nitric oxide and TGF- ⁇ .
  • Agents that are useful in treatment of an inflammatory or autoimmune disorder as disclosed herein such as a substance that binds CIITA and/or modulates e.g. inhibits CIITA binding to a CIITA binding partner, or a substance that modulates e.g. inhibits CIITA production (e.g. RNA with nucleotide sequence complementary to a CIITA gene sequence, which RNA is double- stranded RNA or antisense RNA, or a ribozyme specific for a CIITA gene sequence) can be obtained using routine assay and screening techniques available in the art.
  • a substance that binds CIITA and/or modulates e.g. inhibits CIITA binding to a CIITA binding partner
  • a substance that modulates e.g. inhibits CIITA production e.g. RNA with nucleotide sequence complementary to a CIITA gene sequence, which RNA is double- stranded RNA or antisense RNA, or a ribo
  • the CIITA protein used in the assay may be human, or non-human mammalian, e.g. murine, mouse, rat, rabbit, guinea pig, sheep, goat, cow, pig, cat or dog.
  • reference to CIITA in an assay may be taken to refer to a derivative, variant or analogue of the relevant component which has the requisite, assayable property or activity.
  • test substances may be screened for ability to interact with or bind CIITA e.g. in a yeast two-hybrid system (which requires that both the polypeptide component and the test substance can be expressed in yeast from encoding nucleic acid, see e.g. Evan et al . Mol . Cell . Biol . 5, 3610-3616 (1985); Fields & Song Nature 340, 245-246 (1989) ) .
  • This may for example be used as a coarse screen prior to testing a substance for actual ability to modulate activity.
  • CIITA protein or a fragment thereof may then be tested for ability to affect CIITA activity in a suitable test system, in vitro, ex vivo or in vivo.
  • the substance may be investigated further, in particular for its ability to treat an inflammatory or autoimmune disorder. This may employ a suitable test animal model for the disorder.
  • the agent or substance may be manufactured and/or used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals.
  • the present invention extends in various aspects not only to a substance identified as treating an inflammatory or autoimmune disorder, e.g. ameliorating one or more symptoms, in accordance with what is disclosed herein, but also a pharmaceutical composition, medicament, drug or other composition comprising such a substance, a method comprising administration of such a composition to a patient, e.g. for treatment (which may include preventative treatment) of a disorder, use of such a substance in manufacture of a composition for administration, e.g. for treatment of a disorder, and a method of making a composition comprising admixing such a substance with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients .
  • a substance in accordance with any aspect of the present invention may be formulated in a composition.
  • a composition may include, in addition to said substance, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or one or more other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may include a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
  • administration is preferably in a polypeptide, peptide, nucleic acid molecule, small molecule or other pharmaceutically useful compound according to the present invention that is to be given to an individual, administration is preferably in a
  • prophylactically effective amount or a “therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual.
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of .the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed) , 1980.
  • Targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibody or cell specific ligands. Targeting may be desirable for a variety of reasons; for example if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.
  • these agents may be produced in the target cells by expression from an encoding gene introduced into the cells.
  • the vector may be targeted to the specific cells to be treated, or it may contain regulatory elements that are switched on more or less selectively by the target cells.
  • the agent may be administered in a precursor form, for conversion to the active form by an activating agent produced in, or targeted to, the cells to be treated.
  • a composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • Assays may be conveniently carried out in a 96-well microtitre plate format.
  • Reagents and materials for generating and measuring an end point are well known in the art and are available commercially. Such reagents and materials may be used by a skilled person in accordance with the manufacturer's instructions as appropriate.
  • Another aspect of the invention provides a method of predicting whether an individual with an inflammatory or immune disorder will respond to CIITA modulatory treatment, e.g. anti-CIITA treatment, the method comprising; determining the presence of a CIITA allele associated with susceptibility to the disorder in a sample obtained from the individual .
  • CIITA modulatory treatment e.g. anti-CIITA treatment
  • Another aspect of the invention provides a method of treating an individual with an inflammatory or autoimmune disorder, comprising: identifying the individual as responsive to CIITA modulatory treatment using a method as described herein, and; administering CITTA modulatory treatment to said individual.
  • Examples of CIITA modulatory treatment may include use of one or more statins.
  • Statins is a group of drugs currently approved for lowering cholesterol as primary prevention of cardiovascular disease and as secondary prevention of AMI. This group of pharmacological agents acts by blocking cholesterol synthesis through interference with the activity of 3-hydroxy-3-methylglutaryl co-enzyme A (HMG-CoA) reductase in the mevalonate pathway (Waldman and Kritharides, 2003) .
  • HMG-CoA 3-hydroxy-3-methylglutaryl co-enzyme A
  • the cholesterol lowering effect is mediated by targeting of HMG-CoA reductase in the liver.
  • mevalonate is present in all cells and the down-stream pathway gives rise to numerous metabolites that are active in a range of cellular processes including metabolism, growth regulation and cell to cell signalling.
  • statins have been shown to have anti-inflammatory effects that are not related to cholesterol, e.g. effects on nitric oxide synthase, cytokine and chemokine levels, adhesion molecules and matrix metalloproteinases (Mach, 2002) .
  • the exact molecular pathway (s) operable for this regulation is currently unknown. It has also been demonstrated that statins reduce MHC class II expression and have a direct effect on the transcriptional regulation of the CIITA gene, since the statins Atorvastatin, Lovastatin and Pravastatin inhibit the
  • IFN ⁇ -inducible CIITA IV promoter Kwak et al . , 2000.
  • the exact mechanis (s) for this inhibition remains to be elucidated (Mach, 2002) .
  • the present invention show that genetic polymorphisms in the CIITA gene leading to a reduced induction of transcription of CIITA and MHC molecules upon inflammatory stimuli are associated with inflammatory disease such as AMI, MS and RA, thus, providing a rationale for establishing the genotype of MHC2TA or CIITA protein levels in individuals treated with statins for these conditions (which may include prevention)., potentially regardless of cholesterol levels.
  • the pharmacodynamic profile of currently used statins has been optimized for inhibition of HMG-CoA reductase in the liver.
  • the present invention now singles out CIITA as a direct therapeutic target among the pleiotropic effects of statins, so that monitoring of CIITA levels in appropriate bodily compartments can be used as a mean of optimizing therapy with statins in inflammatory diseases such as AMI, MS and RA. Further optimization of statin treatment in these contexts may also be aimed at blocking the constitutive expression of CIITA.
  • Reference sequences are shown in Table 2.
  • the 2 SNPs in the coding sequence of CIITA I exon 1 are synonymous, i.e. do not result in a changed amino acid sequence in the translated protein.
  • the two haplotypes segregate between the high versus low MHC II responder strains, with the G/T/T/G/G/T haplotype being shared by the three high responders and the T/C/C/A/A/C shared by the three low-responder strains.
  • the functionally relevant polymorphism (s) may be one or several of the ones identified, or may be coupled to the haplotype through linkage disequilibrium.
  • the presence of two distinct haplotypes in the six different strains studied here argues for an evolutionary conserved variation in the rat CIITA locus that is strongly linked to the level of MHC II expression.
  • a large F2 cross where a susceptible and a resistant strain were intercrossed and all individual rats underwent surgery in the VRA paradigm, was performed. Subsequently, all rats were genotyped using approximately 150 microsatellite markers dispersed throughout the genome. This led to the identification of a genomic locus outside of the MHC complex displaying extremely high linkage (LOD score 24) to expression of MHC II in the nervous system. Further fine mapping was performed with additional polymorphic markers. The size of the locus is approximately 20cM. In addition, a smaller influence by the MHC complex itself was detected.
  • the corresponding syntenic human region to our MHC II QTL turn up as "hot spots" in several linkage studies for autoimmune diseases, including insulin-dependent diabetes mellitus (IDDM), rheumatoid arthritis (RA) and MS.
  • IDDM insulin-dependent diabetes mellitus
  • RA rheumatoid arthritis
  • MS MS.
  • VRA was performed in parental rats of each strain and 126 F8 animals.
  • the VRA-induced expression of MHC class II was determined with immunohistochemistry using the OX-6 monoclonal antibody.
  • OX-6 positive cells were present in the grey matter of the ventral horn of the lesioned side as well as in the ventral white matter of the spinal cord that contains crossing motor axons .
  • Measurements of the percentage of positive labeled area in the parental strains demonstrated an almost 5 times greater labeled area in the DA strain.
  • a 4 cM broad VRA4 peak on chr 10 has been identified in a prior F2 cross (Lidman et al . , 2003).
  • CIITA and Ii invariant chain
  • levels of CIITA and Ii mRNAs were determined with real-time RT-PCR in order to explore if strain-specific differences in MHC class II expression could be associated with a differential expression of CIITA.
  • the expression pattern in spleen 3 h after systemic LPS administration was determined in DA and PVG rats. This showed higher expression of CIITA mRNA levels in DA rats compared to PVG.
  • CIITA expression was analyzed in the spinal cord of a group of F8 animals stratified for genotype with the polymorphic marker (D10Mgh25) located in one of the introns of the Mhc2ta gene.
  • the microsatellite-based haplotype map at the Vra4 locus consisting of 15 micro satellite markers across 5.8 Mb at the very acrocentric end of chromosome 10, demonstrated a distinct segregation in the DA, BN, and LEW strains ("high-responders") compared to the E3, PVG, and ACI strains ( ⁇ low-responders”) between the markers AU048079 and D10Mitl4, a region of ⁇ 2.7 Mb containing 21 genes annotated in man, mouse, and/or rat.
  • the Mhc2ta gene as well five of the surrounding genes were sequenced in the DA and PVG.1AV1 strains.
  • Polymorphic loci were further sequenced in additional strains (BN, LEW, E3, ACI) .
  • Seven single nucleotide polymorphisms (SNPs) were identified in the Mhc2ta gene, of which the 6 SNPs located in the promoter region of CIITA defined two haplotypes shared by DA/LEW/BN rats, and PVG/E3/ACI rats, respectively.
  • Three SNPs in the coding sequence were synonymous, while four were located in the promoter regions of the CIITA type III and IV isoforms (position -950 / -683 and -779 / -97, respectively) .
  • SNPs in other genes did not match the pattern of two groups of rats with high and low MHC class II expression, respectively, with the exception of Snn located outside the recombination interval as determined by expression of CIITA and Ii in F10 intra- Vra4 recombinants, and a SNP in the
  • Nubpl 5 'flanking region of Nubpl .
  • Expression analysis of Nubpl in the spinal cord showed no difference in transcript levels between na ⁇ ve DA and PVG animals or 7 and 14 days after nerve injury.
  • the ACI genotype in the exon 10 of the CIITA common region excludes this part of the gene as well as Dexi being the next close gene.
  • the DA(RTl avl ) strain was originally generously provided by Professor Hans Hedrich (Medizinische Hochhoff, Hannover, Germany), while the PVG.1AV1 strain was obtained from Harlan UK Ltd (Blackthorn, UK) .
  • the rats used for experiments were bred at the in-house breeding facility under specific pathogen-free and climate-controlled conditions with 12 h light/dark cycles, housed in polystyrene cages containing wood shavings, and fed standard rodent chow and water ad libitum.
  • the AIL was developed by consecutive crossings of offspring from two pairs of founder rats (male DA and female PVG.1AV1 and vice versa) .
  • the VRA experiment consisted of 24 parental rats and 126 male rats in the F8 generation.
  • Immunohistochemistry and Image analysis The protocol used for immunohistochemistry has been described in detail previously (Lundberg et al . , 2001).
  • Anti-rat la antigen MHC class II; clone OX-6, mouse IgGl, Serotec, Oxford, UK
  • Cy3- conjugated donkey anti-mouse Jackson ImmunoResearch
  • Measurements of the immunofluorescence labeling for MHC class II were conducted using a computer-based image analysis system (Lundberg et al . , 2001). Measurements were recorded blindly on coded slides, with identical computer and camera settings for all sections. Each reading was taken from a 0.24x0.38mm rectangle placed in the ventro-lateral part of the ventral horn with a total of ten different sections analyzed from each animal .
  • RNA samples were isolated from homogenized tissues or cell cultures, respectively, using Qiagen total RNA extraction kit (Hilden, Germany) . Each spinal cord sample consisted of the ipsilateral ventral quadrant from the L3 segment. RNA samples underwent DNase digestion prior to cDNA synthesis in order to avoid amplification of genomic DNA. Reverse transcription was performed with 10 ⁇ l total RNA, random hexamer primers (O.l ⁇ g; Gibco BRL) and Superscript Reverse Transcriptase (200U; Gibco BRL) .
  • Amplification was performed on an ABI PRISM 7700 Sequence Detection System (Perkin Elmer, Norwalk, CT) with a two-step PCR protocol (95°C for 10 min followed by 40 cycles of 95 °C for 15 sec and 60 °C for 1 min.) .
  • PCR-primers for polymorphic simple sequence length polymorphisms were selected from available Internet databases (Rat Genome Database (http://rgd.mcw.edu findable using any web browser), Center
  • genomic DNA was amplified with a standard PCR protocol, and the amplified fragments where separated on 6 % polyacrylamide gels. Genotypes were recorded manually from autoradiograhic films independently by two investigators . DNA from DA and PVG rats were included for
  • Linkage analysis was performed using the MAPMAKER/QTL (Lander and Botstein, 1989) computer program. Image analysis data for MHC class II expression were subjected to log (base 10) transformation in order to obtain more symmetric distribution. Strain comparisons were performed by Student's t-test (IFN- ⁇ and LPS injections) and non-parametric statistics (analysis of CIITA pill and Ii mRNA expression in F8 rats) using GraphPad Prism 3.0 (GraphPad Software Inc, San Diego, CA) . Correlation between expression of CIITA pill and Ii was assed by Spearman rank test.
  • Amplification for sequencing templates was performed on a PTC- 225 (MJ research, Waltham, MA) thermal cycler using a standard three-step PCR protocol with primers purchased from PR0LIG0 (Paris, France) .
  • the sequencing reaction was performed using the BigDye terminator (Applied Biosystems, Foster City, CA) , and separated and recorded on an ABI 3100 (Applied Biosystems, Foster City, CA) .
  • DNA sequences were analyzed using Vector NTI software (InforMax, Frederick, MD) .
  • the complete coding sequence (cds) and 1 kb up stream of Mhc2ta were sequenced in each of the three isoforms' 5 'flanking regions.
  • This gene was identified on the basis of cloning of a gene with a null mutation in hereditary MHC class II deficiency (Bare lymphocyte syndrome; BLS) (Steimle et al., 1993) and has been described as a global regulator of the expression of genes involved in antigen presentation and processing, including MHC class II, invariant chain (la) , HLA-DM, and MHC class I (Chang and Flavell, 1995; Kern et al., 1995; Chang et al . , 1996; Nagarajan et al . , 2002). It does not bind DNA by itself, but rather acts as a platform or chaperone for the assembly of several smaller transcription factors.
  • BLS hereditary MHC class II deficiency
  • Each promoter is activated in a tissue specific manner and lead to the generation of different isoforms of CIITA.
  • Type I and III are important for constitutive MHC class II expression in dendritic cells and B cells, respectively, whereas type IV is important for inducible MHC class II expression in a variety of tissues.
  • an IFN- ⁇ inducible effect on the pill isoform has also been observed.
  • a strong chain of evidence links CIITA to processes of fundamental importance for the immune system.
  • the expression of CIITA is rate limiting for constitutive and IFN- ⁇ -induced MHC class II expression (Otten et al., 1998) and transfection with a CIITA construct bypass the need of an IFN- ⁇ signal for MHC class II expression (Chin et al .
  • CIITA deficient mice are totally devoid of MHC class II expressing cells except of a subset of thymic epithelial cells (Chang et al . , 1996). More subtle differences in the transcriptional activity of CIITA may also have important immunological effects as levels of MHC class II has been shown to modulate immune response (Baumgart et al., 1998) and ectopic expression of CIITA in a transgenic mouse model leads to preferential differentiation into Th2 cells upon TCR activation (Otten et al . , 2003). It has also been shown that several infectious pathogens targets IFN- ⁇ induced expression of CIITA in order to escape the immune system (Hegde et al., 2003).
  • MS Multiple sclerosis
  • EAE Experimental autoimmune encephalomyelitis
  • Interaction between two loci may be studied by a two- dimensional genome scan with a two-QTL model. For every combination of positions, a joint and an interaction LOD score are calculated.
  • the joint LOD (LODj) is a measure of the additive effect of the two loci, whereas the interaction LOD (LODi) addresses epistasis.
  • the CIITA locus previously identified to govern MHC class II expression in the ventral root avulsion (VRA) model was shown to significantly interact with a locus on chromosome 12 harboring the EAE regulating Ncf-1 gene.
  • the CIITA locus thus, exhibits a clinical effect in a rat model for MS.
  • a total of 1068 MOG immunized F7 rats were examined for clinical EAE signs for 31 days post - immunization.
  • Clinical signs defined as a minimum of 2 days at score 1 (tail paralysis) or higher were recorded in 14.8% of animals (158/1068) .
  • Affected animals displayed different degrees of severity and a variety of disease courses (i.e. monophasic, relapsing - remitting and chronic progressive EAE) .
  • the CIITA locus was covered by 6 microsatellite markers, with one marker located within the CIITA gene itself.
  • a genome scan in the F7 EAE revealed linkage to antibody isotype IgG2c, p ⁇ 0.05 for the intra-CIITA marker, with lower levels of IgG2c expressed by animals homozygous for the PVG CIITA allele. There was, however, no significant single QTL correlation to clinical EAE parameters.
  • Dl2Rat23 is located approximately 5 Mb upstream of the Ncf-1 gene
  • D10Arb26 is located between 0.1-0.4 Mb downstream of the CIITA gene. Given the close proximity of DlOArb26, these results strongly suggest CIITA as an important regulator of EAE given interaction with the cl2 locus, possibly through Ncf-1.
  • the MHC complex is the only identified gene region so far displaying significant linkage to MS. It also has a dominant regulatory effect in animal models of EAE. Although this region contains many immunologically relevant genes, the experiments in intra-MHC recombinant strains suggest that mainly the MHC class II region mediates this effect, underscoring the importance of antigen presentation in MS/EAE.
  • the efficiency of antigen presentation does not solely depend upon the amount of MHC class II transcripts (regulated by CIITA) , but also on antigen processing and peptide loading. On theoretical grounds, Ncf-1 is likely to be involved in such activities. However, experiments that prove this notion have yet to be performed. An interactive effect between CIITA governing MHC class II transcription, and region (s) affecting the antigen processing pathway would therefore be of possible importance in the context of EAE/MS.
  • the advanced intercross line was established from EAE - susceptible DA and -resistant PVG.1AV1 that share the RT.1AV1 MHC haplotype, allowing identification of non - MHC genes.
  • One important reason for choosing the DA/PVG strain combination was to permit dense genotyping, since these strains display a high rate of polymorphic microsatellite markers (-60% according to the Whitehead Institute (http://www- genome.wi.mit.edu/rat/public/ findable using any web browser).
  • To create the FI generation two breeding pairs with DA female founders and two breeding pairs with PVG.1AV1 female founders were bred.
  • the F2 generation was produced from seven couples each of FI rats with DA and PVG.1AV1 as female founders, respectively.
  • the F3 generation originated from 50 breeding couples with both types of female founders . Random breeding of 50 males and females produced all following generations except that brother - sister mating was avoided throughout the breeding program. Finally, three F7 litters were produced for MOG - EAE experiments. The litters were similar in size and with almost equal numbers of females and males. 1083 F7 animals were selected for EAE experiments (15 animals died after anesthesia either during immunization or blood sampling procedure) . Inbred DA (Dark Agouti) and PVG.1AV1 rats were originally obtained from Gottinstitut for remediesstierzucht (Hannover, Germany) .
  • Rats were bred and kept at the Karolinska Hospital (Stockholm, Sweden) in a 12h light/dark cycle, housed in polystyrene cages containing aspen wood shavings and had free access to water and standard rodent chow. They were routinely tested for specific pathogens according to a health - monitoring program for rats at the National Veterinary Institute in Uppsala, Sweden. The local ethical committee in Northern Sweden approved the experiments.
  • Rats between 8-11 weeks of age were anaesthetized with halothane and immunized intradermally in the tail base.
  • Each rat received 200 ⁇ l inoculum containing 20 (4:e) 50 (1-2: a) ⁇ g recombinant MOG (aal-125) and 200 ⁇ g Mycobacteria tuberculosis, mixed with 100 ⁇ l incomplete Freund' s adjuvant (Sigma) and 100 ⁇ l phosphate buffered-saline (Life Technologies) .
  • Animals were weighed and clinical signs of disease evaluated from day 7 to day 30-35 p . i . The signs were scored as follows: 1; tail weakness or tail paralysis.
  • Serum was sampled from each rat day 12 p . i .
  • Anti-MOG IgG, IgGl, IgG2a, IgG2b and IgG2c for each rat was determined by ELISA as described.
  • ELISA plates (Nunc, Roskilde, Denmark) were coated with lOO ⁇ l recombinant rat MOG (amino acids 1-125) diluted in 0.1 M NaHC0 3 pH 8.2 to a concentration of 10 ⁇ g/ml. The coated plates were stored overnight at 4°C.
  • the sera for measuring IgG, IgG2a and IgG2b-isotype levels were diluted 1:2000-1:2500 and the sera for IgGl and IgG2c were diluted 1:200-1:250.
  • Antiserum was diluted as follows: IgG, IgG2a and IgG2b 1:2000; IgGl, 1:1000; IgG2c, 1:500 (Nordic).
  • Goat-anti- rabbit conjugate was diluted 1: 10000 (Nordic).
  • OD values were read at 450 nm.
  • Each plate had DA serum (immunized with MOG) as a positive control in duplicates. Arbitrary units were calculated for each rat and for each IgG isotype, by comparing the values with the standard curve of the positive control, the DA, for each ELISA plate.
  • Genotyping and statistical analysis A total of 151 clinically affected rats and 162 randomly selected unaffected rats were genotyped. Genotyping was performed using standard microsatellites (GENSET, Paris, France) with primer radioactive labeling and fragment analysis by size. Genotype analyses for the SNP markers were run using the Pyrosequencing methodology (and confirmed by sequencing using the ABI Prism 3100) . Affected animals were selected on the basis of displaying unambiguous signs of the disease (minimum score 1 for more then 2 days accompanied with weight loss) . Rats in the unaffected group did not display any signs of disease, including a steady increase in weight. DNA was extracted from the tail tip according to a standard protocol ⁇ Laird, 1991 #6143 ⁇ .
  • the R5 recombinant and adjacent regions of approximately 30 cM were genotyped with fifty - five microsatellite markers ( ⁇ 1 cM spacing in average) microsatellite markers that were utilized for genotyping (GENSET, Paris, France) .
  • the RA cohort
  • the MS cohort consisted of 550 subjects (412 females and 138 males; age 13-
  • Genotype was determined with Taqman PCR in both RA and AMI.
  • DASH was performed as previously described by DASH, 5'nuclease assay or by MALDI .
  • Genotype and allele frequencies were compared using the Chi- square test or Fisher exact test and differences were considered significant at p ⁇ 0.05 (StatView, version 5.0, SAS Institute Inc., USA). Haplotype frequencies were estimated by the EM algorithm using the Arlequin software 2.000 nimals and breeding
  • Peripheral blood samples were obtained from RA patients with GGG and AGG haplotypes. Peripheral blood was sampled in heparinized Lymphoprep tubes (Vacutainer CPT, Becton Dickinson and Company, USA) . Peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation. Cells from interphase were collected, washed 2 times with Dulbecco's phosphate-buffered saline (PBS) . The proportion of viable cells was assessed with trypan blue. More than 95% of the cells excluded trypan blue in each preparation. The stimulation protocol was titred in initial experiments using PBMC from three healthy subjects. Immediately after preparation, 1, 10 or 50 units of recombinant human IFN- ⁇ (Preprotech) , or medium alone, was added to the cultures. After 6hrs cell were lysed and total RNA extracted.
  • PBMC Peripheral blood mononuclear cells

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Abstract

L'invention concerne l'association d'allèles du gène mhc2ta ou CIITA à la sensibilité à une maladie ; des méthodes de détermination de la sensibilité chez des individus ; des analyses d'agents thérapeutiques ; et des traitements, en particulier associés à des troubles inflammatoires et auto-immuns, de type infarctus du myocarde aigu, polyarthrite rhumatoïde et sclérose en plaques.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0648836A1 (fr) * 1993-08-26 1995-04-19 Bernard François Prof. Mach Transactivateur du complexe majeur d'histomcompatibilité de la classe II et ses utilisations
US5672473A (en) * 1994-08-24 1997-09-30 President And Fellows Of Harvard College Methods of identifying compounds useful for treating autoimmune diseases
WO2001066800A2 (fr) * 2000-03-07 2001-09-13 Whitehead Institute For Biomedical Research Polymorphismes humains a nucleotide unique
US20020151691A1 (en) * 1997-04-22 2002-10-17 Bernard Mach Nucleic acid sequences of ciita genes which can be involved in controlling and regulating the expression of genes encoding mhc type ii molecules, and their use, in particular as drugs.

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0648836A1 (fr) * 1993-08-26 1995-04-19 Bernard François Prof. Mach Transactivateur du complexe majeur d'histomcompatibilité de la classe II et ses utilisations
US5672473A (en) * 1994-08-24 1997-09-30 President And Fellows Of Harvard College Methods of identifying compounds useful for treating autoimmune diseases
US20020151691A1 (en) * 1997-04-22 2002-10-17 Bernard Mach Nucleic acid sequences of ciita genes which can be involved in controlling and regulating the expression of genes encoding mhc type ii molecules, and their use, in particular as drugs.
WO2001066800A2 (fr) * 2000-03-07 2001-09-13 Whitehead Institute For Biomedical Research Polymorphismes humains a nucleotide unique

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BONTRON S ET AL: "Two novel mutations in the MHC class II transactivator CIITA in a second patient from MHC class II deficiency complementation group A." HUMAN GENETICS. APR 1997, vol. 99, no. 4, April 1997 (1997-04), pages 541-546, XP002322239 ISSN: 0340-6717 *
DZIEMBOWSKA MAGDA ET AL: "Three novel mutations of the CIITA gene in MHC class II-deficient patients with a severe immunodeficiency." IMMUNOGENETICS. FEB 2002, vol. 53, no. 10-11, February 2002 (2002-02), pages 821-829, XP002322240 ISSN: 0093-7711 *
RASMUSSEN H B ET AL: "Genetic susceptibility to multiple sclerosis: detection of polymorphic nucleotides and an intron in the 3' untranslated region of the major histocompatibility complex class II transactivator gene." HUMAN IMMUNOLOGY. APR 2001, vol. 62, no. 4, April 2001 (2001-04), pages 371-377, XP002322238 ISSN: 0198-8859 cited in the application *
SARTORIS SILVIA ET AL: "Analysis of CIITA encoding AIR-1 gene promoters in insulin-dependent diabetes mellitus and rheumatoid arthritis patients from the Northeast of Italy: Absence of sequence variability" HUMAN IMMUNOLOGY, vol. 61, no. 6, June 2000 (2000-06), pages 599-604, XP002322241 ISSN: 0198-8859 *
STEIMLE V ET AL: "COMPLEMENTATION CLONING OF AN MHC CLASS II TRANSACTIVATOR MUTATED IN HEREDITARY MHC CLASS II DEFICIENCY (OR BARE LYMPHOCYTE SYNDROME)" CELL, CELL PRESS, CAMBRIDGE, NA, US, vol. 75, no. 1, 8 October 1993 (1993-10-08), pages 135-146, XP002051559 ISSN: 0092-8674 *

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