WO1996030511A1

WO1996030511A1 - Polymorphic and multicopy members of mhc gene families

Info

Publication number: WO1996030511A1
Application number: PCT/AU1996/000184
Authority: WO
Inventors: Roger Letts Dawkins
Original assignee: The Immunogenetics Research Foundation Incorporated
Priority date: 1995-03-29
Filing date: 1996-03-29
Publication date: 1996-10-03

Abstract

A nucleotide sequence coding for a gene relevant to an MHC associated disease, or gene sequence substantially homologous therewith, derivative, mutant or fragment thereof, said sequences being haplotype specific, highly polymorphic and duplicated within the MHC.

Description

POLYMORPHIC AND MULTICOPY MEMBERS OF MHC GENE FAMILIES

The present invention relates generally to DNA sequences related to the major histocompatibility complex (MHC). In particular, the present invention relates to isolation, identification, and characterization of nucleotide sequences of the MHC which are implicated in MHC associated diseases including autoimmune diseases and immunoregulatory diseases. The present invention also contemplates the use of the nucleotide sequences, and functionally active parts, fragments, derivatives and/or analogues thereof, for expression of peptides, and compositions including same for use in prevention, treatment, diagnosis and/or prophylaxis of MHC associated diseases. The present invention further contemplates methods of preventing, treating or otherwise ameliorating MHC associated diseases.

The term "MHC associated diseases" as used herein refers to any condition which involves an interaction or otherwise an association with the major histocompatibility complex either directly or indirectly and of which the MHC is a component of the pathology.

The MHC is a cluster of genes encoding the major histocompatibility antigens, some complement proteins and other surface proteins of immune system cells. In humans, it is known as the HLA complex and in mice the H-2 complex.

The MHC has been implicated in the aetiology of numerous diseases including immunological, neurological and infectious disorders, as well as diseases characterized by abnormal cell growth and development such as cancers of various tissues and organs, [Tiwari, J.L. & Terasaki, P.I. (1985) HLA antigens associated with diseases. In: HLA and Disease Associations (ed. by J.K. Tiwari & P.I. Terasaki), p. 42, New York, Springer-Verlag]. The nature of these associations is, in most cases, not understood. Associations with MHC ancestral haplotypes (AH) rather than single alleles suggest that disease susceptibility may be the result of cis interactions between existing genes or the result of alleles of unidentified MHC genes carried by particular haplotypes.

The MHC appears to be rich in coding sequences with an average of at least one gene in every 40 kb [Marshall, B., Leelaywat, C, Degli-Esposti, M.A. Pinelli, M., Abraham, LJ. and Dawkins, R.L (1993) New MHC genes. Hum Immunol 38: 24-29]. Although some regions have a high density of loci (ca. one gene every 15-20kb), such as the region between G18 and BAT1 , for a long time the region between BAT1 and HLA-B (approximately 170 kb) remained apparently devoid of genes. A study in this region indicated that highly polymorphic and duplicated genomic segments were present [Leelayuwat, C, Abraham, L.J., Tabarias, H., Christiansen, F . and Dawkins, R.L. (1992) Genomic organisation of a polymorphic duplicated region centromeric of HLA-B. Immunogenetics 36: 208-212]. Recently some transcripts that map to the region were identified [Marshall, B., Leelayuwat, C, Abraham, L.J., Pinelli, M. and Dawkins, R.L. (1993) Large transcripts and sequence from a polymorphic MHC region of 170kb implicated in susceptibility to autoimmune disease. Immunogenetics 39: 15-20].

The Applicant has previously established strategies to identify genes susceptible to MHC associated diseases. Whilst the Applicant does not wish to be restricted by theory, the strategies were based on the following hypotheses: (i) MHC disease associations are accounted for by unidentified genes, (ii) the relevant genes are polymorphic and/or duplicated, (iii) haplotype-specific polymorphisms result in qualitative and/or quantitative differences in gene expression when comparing disease and non-disease associated haplotypes.

Using these strategies, the Applicant has identified further novel polymoφhic sequences related to MHC associated diseases. Preferably, the gene sequences are highly polymoφhic and duplicated in conditions which are

MHC associated. The region of the MHC from which these DNA sequences may be derived is rich in genes important for susceptibility to MHC associated diseases.

A number of genes critical to the aetiology of MHC associated diseases have been identified. The sequences were analysed for coding potential and their relevance assessed by taking account of the pattern of polymoφhism when ancestral haplotypes were compared. These genes otherwise known as susceptibility genes on MHC haplotypes are implicated as being associated with MHC diseases.

Antibodies to TNF gene products from the MHC are said to have beneficial effects in relation to diseases including cerebral malaria and rheumatoid arthritis. However, the antibodies available have not found widespread application to date with respect to all members of the TNF family. An effective antibody against TNF genes would need to react with all of the different members of the TNF related family within the MHC. Antibodies found in the prior art therefore are reactive to only some members of the TNF family.

It would therefore be desirable to identify those regions common to all members of an MHC gene family which could be used to identify and be indicative of that gene family and of a specific disease condition.

One gene family, designated as PERB11 , has been identified [Leelayuwat, C. et al; "A new polymoφhic and multi-copy MHC gene family related to non- mammalian class 1"; Immunogenetics, (1994); 40; 339-351].

The PERB11 gene family belongs to the immunoglobulin supergene family and is closely related to MHC class 1 neonatal IgG Fc receptor (FcRn), and Zn- alpha-2 glycoprotein, all of which appear to be peptide/protein binding receptors. The PERB 11 is polymoφhic at the DNA and protein levels and it has been noted that multiple copies occur in the MHC. Hence, it would be desirable to be able to identify and locate specific sequences characteristic of the PERB11 gene family for characterization of the gene products and for typing puφoses which associate the PERB 11 gene family to specific disease conditions preferably relating to MHC associated diseases. It would also be desirable to provide specific sequences for regulating immune responses such as those which lead to Myasthenia gravis and IgA production. Knowledge of these specific sequences will be beneficial in treating such diseases including IgA deficiencies. Typing for PERB11 gene family members will help in the identification of MHC ancestral haplotypes.

It is an object of the present invention to overcome or at least alleviate some problems of the prior art.

Accordingly in first aspect of the present invention there is provided a nucleotide sequence coding for a gene relevant to an MHC associated disease, or gene sequence substantially homologous therewith, derivative, mutant or fragment thereof, said sequences being haplotype specific, highly polymoφhic and duplicated in the MHC.

In a preferred aspect of the present invention there is provided a nucleotide sequence coding for a gene relevant to an MHC associated disease, or gene sequence substantially homologous therewith, derivative, mutant or fragment thereof, said sequences being haplotype specific, highly polymoφhic and duplicated within a distinct gene family of the MHC.

The MHC contains susceptibility genes for numerous autoimmune diseases and a number of gene families which are polymoφhic with multiple copies exist on the MHC. It has been found that distinct sequences within the gene families show substantial homology and may be used to identify a number of members of a related gene family.

Preferably the gene family is the PERB11 gene family or the TNF gene family. Accordingly, in a preferred aspect of the present invention there is provided a nucleotide sequence of an MHC associated gene sequence of a PERB11 gene family having a nucleotide sequence as illustrated in Figure 1 or sequence substantially homologous therewith, active part, derivative, mutant, analogue or fragment thereof. Preferably the nucleotide sequence is from nucleotide 7687 to 7944 or from nucleotide 8218 to 8359 as illustrated in Figure 1 or sequence substantially homologous therewith, active part, derivative, mutant, analogue or fragment thereof.

Within the PERB11 gene family it has been found that there are regions of the sequence which are characteristic of the PERB11 gene family. In a further preferred embodiment of the present invention there is provided a nucleotide sequence corresponding to a region having a nucleotide sequence from nucleotide 7751 to 7944 or nucleotide 8218 to 8343 as illustrated in Figure 1 or sequence substantially homologous therewith, active part, derivative, mutant or fragment thereof.

Within the regions are nucleotide sequences which more specifically characterize PERB11 gene families. More preferably the nucleotide sequences characteristic of the PERB11 gene family are those sequences having a nucleotide sequence from nucleotide 7793 to 7825, or nucleotide 7857 to 7889 or nucleotide 8254 to 8298 as illustrated in Figure 1 or sequence substantially homologous therewith, active part, derivative, mutant or fragment thereof.

In a further preferred aspect of the present invention, there is provided a nucleotide sequence of an MHC associated gene sequence of a TNF-related gene-family, said nucleotide sequence having the sequence substantially as illustrated in Figure 25 or sequence substantially homologous therewith, fragment, derivative, mutant and/or analogue thereof. TNF is a cytokine involved in many regulatory activities of T and B cell regulation. TNF is synthesized in response to insults by a variety of cell types including both hemotopoietic and non-haematopoietic cells and is generally regarded as one of the primary initiating events in the inflammatory cascade.

Applicants have found a novel member of the TNF family which shows substantial homology with TNF and which may be used to identify a number of members of the TNF-related gene family which are not reactive to TNF antibodies or other members of the TNF family.

The nucleotide sequence may be found in the MHC region between the HLA-B and the TNF loci, preferably in the CL region. These sequences are implicated in MHC associated diseases and preferably related to susceptibility genes for MHC associated diseases.

In a preferred embodiment of the present invention, there is provided a region of homology between the nucleotide sequence of the TNF-related gene family sequence and TNF, said homologous nucleotide sequence having a homologous nucleotide sequence corresponding to the sequence in Figure 26 or sequence substantially homologous therewith, fragment, derivative, mutant and/or analogue thereof.

Substantial homology exists between the nucleotide sequence of the present invention and TNF. These regions of homology are believed to be functionally important in MHC associated diseases. Similar regions have been identified in other TNF-related molecules (Browning et. al., (1993); Cell; 72; 847-

856).

In a further preferred embodiment, there is provided a nucleotide sequence of a TNF-related gene family or sequence substantially homologous therewith, active part, derivative, mutant, fragment and/or analogue thereof, said nucleotide sequence corresponding to the sequence from nucleotide 341 to 367, nucleotide 383 to 394 or nucleotide 578 to 610 substantially as illustrated in Figure 27 or a sequence substantially homologous therewith, active part, derivative, mutant, fragment and/or anologue thereof.

In a further aspect of the present invention there is provided an amino acid sequence of a PERB11 gene family encoded by a nucleotide sequence as illustrated in Figure 1 or a sequence substantially homologous therewith, active part, derivative, mutant, analogue or fragment thereof. Preferably the nucleotide sequence is from nucleotide 7687 to 7944 or from nucleotide 8218 to 8359 as illustrated in Figure 1 or sequence substantially homologous sequence, active part, derivative, mutant or fragment thereof. Preferably, the amino acid sequence of the PERB11 gene family is encoded by a nucleotide sequence corresponding to a region having a nucleotide sequence from nucleotide 7751 to 7944 or nucleotide 8218 to 8343 as illustrated in Figure 1 or a sequence substantially homologous therewith, active part, derivative, mutant or fragment thereof. More preferably the amino acid sequence of the PERB11 gene family is encoded by a nucleotide sequence from nucleotide 7793 to 7825 or nucleotide 7857 to 7889 or nucleotide 8254 to 8298 as illustrated in Figure 1 or sequence substantially homologous therewith, active part, derivative, mutant or fragment thereof. It is also preferable that the amino acid sequence of a PERB11 gene family is as illustrated in Figure 2. Most preferably the amino acid sequence is YDRQKCRAKPQ, KTWDRETROLT or IHEDNSTRSSQHFYY or a sequence substantially homologous therewith, active part, derivative, mutant or fragment thereof.

In a further aspect of the present invention, there is provided a TNF-related gene family amino acid sequence encoded by a nucleotide sequence of Figure 25 or sequence substantially homologous therewith, derivative, mutant or fragment thereof. Preferably the TNF-related gene family amino acid sequence is located between the HLA B and the TNF- loci of the MHC , preferably in the CL region of the MHC. More preferably the amino acid sequence of a TNF-related gene family is encoded by the nucleotide sequence of Figure 26 or sequence substantially homologous therewith, derivative, mutant or fragment thereof.

In a further preferred form, the amino acid sequence of a TNF-related gene family is encoded by a nucleotide sequence corresponding to the sequence from nucleotide 341 to 367, nucleotide 383 to 394 or nucleotide 578 to 610 substantially as illustrated in Figure 27 or a substantially homologous sequence, active part, derivative, mutant, fragment and/or analogue thereof. Even more preferred, the amino acid sequence is encoded by a nucleotide sequence as illustrated in Figure 28 and is designated CL.6.5.40. Most preferably the amino acid sequence of a TNF-related gene family is encoded by an amino acid sequence NLAHLVGDAL, WQWL or GIYSLKGKCSF respectively or a sequence substantially homologous therewith, active part, derivative, mutant or fragment thereof.

The nucleotide sequence of a gene relevant to an MHC associated disease according to the present invention may be a genomic or a cDNA sequence or sequence substantially homologous therewith, active part, derivative, mutant or fragment thereof.

The cDNA sequences may be obtained by a process including the steps of: providing a source of animal cells; isolating the mRNA from the cells; treating the mRNA to produce cDNA; and probing for cDNA of genes identified as relevant to the aetiology of an MHC associated diseases, said cDNA sequences being haplotype specific polymoφhic and duplicated within a distinct gene family of the MHC.

Preferably the gene family is a PERB11 gene family or a TNF-related gene family. The cells may be animal cells, preferably human cells and more preferably cells of the immune system including macrophages and lymphocytes.

The cells so isolated may be stimulated in vivo to enhance mRNA production. For example, the cells may be stimulated by a mitogen such as pokeweed (Phytolacca americana) also known as pokeweed mitogen (PWM), polyvinylpyrrolidone (PVP), polyadenylicpolyuridylic acid (poly(A-U)), purified protein derivate lipopolysaccharide (LPS), staphylococcal organisms or products thereof, Bacto-streptolysin O reageant (SLO), Staphylococcal phage lysate (SPL), Epstein-Barr virus (EBV), Nocardia water-soluble mitogen (NWEM), phytohemagglutinin (PHA) Concanavalin A (Con A) and dextran-sulphate and mixtures thereof.

mRNA may be isolated by any known method, for example, using an oligo dT column.

The production of cDNA from the mRNA may be undertaken in any suitable manner. A number of techniques are known per se in the art for this production. The cDNA may be probed with a DNA probe for an homologous protein such as TNF-α and TNF-β, TNF related activation protein (TRAP), or fibroblast growth factor receptor 3 (FGFR3).

The probing of cDNA for a gene associated with an MHC associated disease said gene preferably from a PERB11 gene family or the TNF-related gene family, may be conducted using any nucleotide sequence as described above as a probe to detect corresponding cDNA sequences.

Alternatively, amino acid sequences encoded by the nucleotide sequences, as described above, may be used to raise antibodies. The antibody may be polyclonal or monoclonal. These antibodies may then be used as probes in a method familiar to the skilled addressee to identify clones expressing cDNA and encoding products antigenic to the raised antibodies. In a further aspect of the present invention, there is provided, clones which express the nucleotide sequence of a gene relevant to an MHC associated disease, to produce corresponding polypeptides. In a preferred aspect the clone expresses a nucleotide sequence of the PERB11 gene family having a nucleotide sequence as illustrated in Figure 1 or a sequence substantially homologous therewith, active part, derivative, mutant, analogue or fragment thereof. Preferably the nucleotide is from nucleotide 7687 to 7944, or nucleotide 8218 to 8359 as illustrated in Figure 1 or sequence substantially homologous therewith, active part, derivative, mutant or fragment thereof.

More preferably, the clone may express a nucleotide sequence corresponding to a sequence of nucleotide 7751 to 7944 or nucleotide 8218 to 8343 as illustrated in Figure 1 or sequence substantially homologous therewith, active part, derivative, mutant or fragment thereof. Even more preferred the clone may express nucleotide sequences having a nucleotide sequence from nucleotide 7793 to 7825, or nucleotide 7857 to 7889 or nucleotide 8254 to 8298 as illustrated in Figure 1 or sequences substantially homologous therewith, active part, derivative, mutant or fragment thereof. Most preferably the polypeptide encoded by the clone has an amino acid sequence YDRQKCRAKPQ, KTWDRETROLT or IHEDNSTRSSQHFYY or a sequence substantially homologous therewith, active part, derivative, mutant or fragment thereof.

In a further preferred aspect of the present invention the clone expresses a nucleotide sequence of a TNF-related gene family sequence substantially as illustrated in Fig. 25 or a sequence substantially homologous therewith, active part, derivative, mutant or fragment thereof. More preferably the clone expresses a nucleotide sequence illustrated in Figure 26, or sequence substantially homologous therewith, active part, derivative, mutant or fragment thereof. Preferably the sequences expressed by the clone may correspond to areas of homology between the TNF-related sequence and TNF, said sequences having a nucleotide sequence corresponding to the sequence from nucleotide 341 to 367, nucleotide 383 to 394 or nucleotide 578 to 610 substantially as illustrated in Figure 27. More preferably the clone encodes an amino acid sequence encoded by a nucleotide sequence as illustrated in Figure 28 and designated CL.6.5.40. Most preferably the clone encodes an amino acid sequence of a TNF-related gene family having amino acid sequences NLAHLVGDAL, WQWL or GIYSLKGKCSF respectively or a sequence substantially homologous therewith, active part, derivative, mutant or fragment thereof.

The expression may be accomplished by any suitable manner. Standard recombinant techniques may be used including the use of live vectors (for example Vaccinia). Alternatively a recombinant expression vector including DNA sequences may be injected directly into the tissue of a suitable host animal and the peptide directly expressed. More alternatively, the recombinant protein may be expressed directly into a cell culture or recovered by standard methods or expressed in a host cell for protein purification by standard methods known to the skilled addressee.

The cDNA may be assembled into a suitable vector that will enable transcription and the subsequent expression of the cloned DNA, either in prokaryotic hosts (e.g. bacteria) or eukaryotic hosts (e.g. mammalian cells.)

In a preferred aspect, there is provided a recombinant polypeptide encoded by a clone which expresses the nucleotide sequences of a gene relevant to an MHC associated disease, preferably a PERB11 gene family or a TNF related gene family as hereinbefore described.

In an even further aspect of the present invention there is provided an antibody reactive to a polypeptide or native protein relevant to an MHC associated disease, said polypeptide or native protein encoded by a nucleotide sequence being haplotype specific, highly polymoφhic and duplicated. Preferably the nucleotide sequence is within a distinct gene family of the MHC. Preferably, the gene family is PERB11 or TNF-related. ln an even further aspect of the present invention there is provided an antibody reactive to a PERB11 gene family polypeptide or native protein derived from the MHC, said polypeptide or native protein being associated with an MHC disease and encoded by a nucleotide sequence as illustrated in Figure 1 or sequence substantially homologous therewith, active part, derivative, mutant, analogue or fragment thereof. Preferably the sequence is from nucleotide 7687 to 7944 or nucleotide 8218 to 8359 as illustrated in Figure 1 or sequence substantially homologous therewith, active part, derivative, mutant or fragment thereof.

More preferably, the antibody is reactive to the polypeptide or native protein from the alpha 1 or alpha 2 domain of a PERB11 gene family which is preferably encoded by the nucleotide sequence corresponding to nucleotide 7751 to 7944 or nucleotide 8218 to 8343 as illustrated in Fig. 1. Preferably, the antibodies are reactive to polypeptides or native proteins encoded by nucleotide sequences from nucleotide 7793 to 7825, or nucleotide 7857 to 7889 or nucleotide 8254 to 8298 as illustrated in Figure 1 or sequence substantially homologous therewith, active part, derivative, mutant or fragment thereof. Most preferably the antibody is reactive to a polypeptide or native protein having the amino acid sequence YDRQKCRAKPQ, KWDRETRDLT or IHEDNSTRSSQHFYY as illustrated in Figure 3 or sequence substantially homologous therewith, active part, derivative, mutant, analogue or fragment thereof.

The antibody may be monoclonal or polyclonal and prepared by methods familiar in the art.

Accordingly, in a further preferred embodiment, the present invention provides antibodies PB11-4D, PB11-5D and PB11-6D having specificity to peptides corresponding to YDRQKCRAKPQ, KTWDRETRDLT or

IHEDNSTRSSQHFYY respectively or a peptide having a similar tertiary structure/epitope. The invention further includes antibodies having similar staining patterns to those provided by specific antibodies PB11-4D, PB11-5D and PB11-6D and showing similar specificity.

In a further aspect of the present invention, there is provided an antibody reactive to a TNF-related gene family polypeptide or native protein derived from the MHC, said polypeptide or native protein being associated with an MHC disease and encoded by a nucleotide sequence according to Figure 25 or sequence substantially homologous therewith, derivative, mutant or fragment thereof. Preferably the TNF-related gene family antibody is reactive to a polypeptide or native protein encoded by a sequence located between the HLA B and the TNF- loci of the MHC , preferably in the CL region of the MHC. More preferably the antibody of a TNF-related gene family is reactive to a polypeptide or native protein encoded by the nucleotide sequence of Figure 26 or sequence substantially homologous therewith, derivative, mutant or fragment thereof.

In a further preferred form, the antibody of a TNF-related gene family is encoded reactive to a polypeptide or native protein encoded by a nucleotide sequence corresponding to the sequence from nucleotide 341 to 367, nucleotide 383 to 394 or nucleotide 578 to 610 substantially as illustrated in Figure 27 or a substantially homologous sequence, active part, derivative, mutant, fragment and/or analogue thereof. Even more preferred, the antibody is reactive to a polypeptide encoded by a nucleotide sequence as illustrated in Figure 28 and is designated CL.6.5.40. Most preferably the antibody is reactive to a polypeptide or native protein of a TNF-related gene family encoded by an amino acid sequence NLAHLVGDAL, WQWL or GIYSLKGKCSF respectively or a sequence substantially homologous therewith, active part, derivative, mutant or fragment thereof.

The antibody may be monoclonal or polyclonal and prepared by methods familiar in the art. The antibody includes those antibodies which have a similar function having a similar specificity but may not be identical in every respect. ln a further aspect of the present invention there is provided a method for treating or preventing an MHC associated disease in an animal, said method including administering to said animal an effective amount of a polypeptide derived from a nucleotide sequence relevant to an MHC associated disease or sequence substantially homologous therewith, derivatives, mutants or fragments thereof, said sequences being haplotype specific, highly polymoφhic and duplicated within a distinct gene family the MHC.

Preferably the gene family is PERB11 or TNF-related gene family.

By "administering to said animal an effective amount of a polypeptide" is meant administration for a time and under conditions sufficient to prevent, reduce or otherwise ameliorate the MHC associated disease.

Most preferably the MHC associated disease is associated with Psoriasis, Nasopharyngeal carcinomas and Spondyloarthopathies, Myasthenia gravis, IgAd and CVI, cerebral malaria, rheumatoid arthritis, AIDS and cachexia.

A further aspect of the invention provides a method for diagnosing an MHC associated disease in an animal, said method including: providing a sample from a patient suspected of having an MHC associated disease; providing a probe related to an MHC associated disease; probing the sample with the probe; and comparing the sample with a sample derived from a patient which does not have the MHC associated disease.

Most preferably the MHC associated disease is associated with Psoriasis, Nasopharyngeal carcinomas and Spondyloarthopathies, Myasthenia gravis, IgAd and CVI, cerebral malaria, rheumatoid arthritis, AIDS and cachexia. Typing for the MHC associated gene sequences such as those of the PERB11 gene family or TNF-related gene family may be used to identify ancestral haplotypes.

The sample may be any biologically sample such as blood and blood derived products, tissue, saliva, urine or faeces.

The probe is preferably a nucleotide sequence relevant to an MHC associated disease as hereinbefore described or sequence substantially homologous therewith, derivatives, mutants or fragments thereof, said sequence being haplotype specific, highly polymorphic and duplicated within a distinct gene family of the MHC.

The probe may also be an antibody as hereinbefore described reactive to a peptide as hereinbefore described encoded by the nucleotide sequence of a PERB11 gene family or of a TNF-related gene family sequence. The antibody may be monoclonal or polyclonal.

Preferably the probe is PB11-4D, PB11-5D or PB11-6D as hereinbefore described or an antibody having similar specificity or providing similar staining patterns to PB11-4D, PB11-5D or PB11-6D.

For use as a diagnostic agent in detection of MHC associated diseases, the probe may be conjugated to an appropriate diagnostic agent such as a radioactive label to detect the MHC disease.

The probing with a suitable antibody may further include subjecting the product produced thereby to a detection assay. The detection assay may include western blot techniques or directly probing tissue samples. The detection assay may be an immunoprecipitation assay, a radioimmunoassay, an enzyme-linked immunoassay or immunofluorescent assay. ln a further preferred aspect of the preset invention there is provided a method of identifying a protein or polypeptide related to an MHC associated disease including obtaining genomic DNA from an MHC associated disease sample; translating a nucleotide sequence from the genomic DNA coding for a gene relevant to an MHC associated disease or gene sequence substantially homologous therewith, derivative, mutant or fragment thereof, said sequence being haplotype specific highly polymoφhic and duplicated in the MHC; obtaining a polypeptide from the translated nucleotide sequence; raising an antibody to the polypeptide which antibody is produced by a method including challenging an animal with the polypeptide or native protein; harvesting antibodies produced from said animal in the serum or plasma, or from immune cells of the animal; probing an MHC associated or non-MHC associated disease sample with the antibody; comparing patterns of an MHC associated and non-MHC associated disease samples obtained by probing to identify a protein related to an MHC associated disease.

Preferably the genomic DNA is derived from a biological sample including tissues, cells, blood, urine and faeces.

The nucleotide sequence is preferably a sequence from a distinct gene family of the MHC. More preferably, the nucleotide sequence is as hereinbefore described for a PERB11 gene family or a TNF-related gene family.

Translation is by any means familiar to the skilled addressee. Preferably, the translation includes translating the nucleotide sequence so as to obtain all forms of a translated peptide sequence incoφorating a frame shift in the nucleotide sequence. In general, within one frame shift, there will be 6 different peptides translated from any given nucleotide sequence. The polypeptide so obtained following translation may be isolated and purified by any means including protein gel separation, chromatography or affinity chromatography.

Generally, the peptides are used to immunize an animal for instance intravascularly, intramuscularly, intraperitoneally or intradermally. The animal is bled prior to inoculation and after a sufficient time, preferably 4 to 9 weeks post inoculation, the animals are bled to obtain the antibodies. Specificity of the antisera are confirmed by testing sera against all inoculating peptides using standard tests such as an enzyme linked immunosorbent assay (ELISA).

Antibody can be obtained from the serum or plasma or by conventional methods. For example precipitation with ammonium sulphate, fractionation with caprylic acid, ion exchange chromatography or by binding and elution from immobilized protein G or protein A may be suitable supports, e.g. CNBr-activated Sepharose 4B (Pharmacia) Affi-gel (Bio-RAD) or other affinity chromatography supports able to bind proteins.

Application of the antibody to an MHC associated or non-associated disease sample may be by way of any detection means including immunostaining or immunofluorescence on samples including direct tissue samples, blood smears, urine samples, faecal samples, fractional protein, etc.

Patterns of staining may provide an indication of whether an animal is more susceptible to a MHC associated disease by comparing with a sample from a non-MHC associated disease sample.

In a further preferred aspect of the present invention the antibody as hereinbefore described may be used for the affinity purification, preferably immuno-affinity purification of antigen or an MHC associated disease polypeptide, preferably a PERB11 gene family or TNF related gene family peptide. Accordingly in a preferred aspect there is provided a method for purifying an MHC associated disease polypeptide or native protein preferably a PERB11 gene family or a TNF related gene family peptide which method includes providing a protein mixture; an antibody against a polypeptide encoded by a nucleotide sequence relevant to an MHC associated disease said nucleotide sequence being haplotype specific, highly polymoφhic and duplicated with a distinct gene family and immobilized on a suitable support, which antibody is produced by a method including challenging an animal with a peptide encoded by a nucleotide sequence relevant to an MHC associated disease as hereinbefore described; harvesting antibodies produced from said animal in the serum, plasma or from immune cells; subjecting the protein mixture to an affinity chromatography utilizing the immobilized antibody; and isolating the purified polypeptide.

Preferably the gene families are selected from PERB11 gene family or TNF-related gene families.

The protein mixture may be a crude protein mixture obtained from any biological source for example animal cells, tissues, blood, urine and faeces.

Immobilized antibody can then be applied to the fractionation and purification of specific polypeptide from a complex cell or antigen extract by affinity chromatography. After binding of polypeptides to immobilized antibody, unbound macromolecular species can be washed away from the solid support with, for example buffers containing 1.5M NaCl. Subsequently the protein can be eluted from the affinity column with, e.g. low or high pH buffer or buffers containing chaotropic ions, e.g. 0.5 - 3.0 M sodium thiocyanate.

The application of the antibody probe to affinity chromatography enables sufficient quantities of specific antigens or proteins to be rapidly isolated from a complex crude extraction mixture for biochemical characterization, amino-acid sequencing and vaccination of animals. Application of affinity chromatography for obtaining antigen(s) avoids the difficulties often encountered when applying conventional biochemical techniques to the purification of an antigen about which little or no data is known. It also obviates the need to raise polyclonal or monoclonal antibodies for the puφose of "analytical" affinity chromatography. Large scale preparation may however require the preparation of polyclonal or monoclonal antibodies.

The proteins or polypeptides isolated or located may be used in the preparation of monoclonal antibodies. The monoclonal antibodies may form the basis of a passive treatment of the disease discussed above. Having identified the antigen(s) or polypeptides, molecular biology or chemical techniques, e.g. cloning techniques may be used to produce unlimited amounts of this antigen or alternatively synthetic peptides corresponding to different fragments of the identified antigens or peptides may be used as a means to produce a vaccine. ln a further aspect of the present invention there is provided a method for preparing a synthetic antigenic polypeptide relevant to an MHC associated disease which method includes providing a cDNA library, or genomic library derived from a sample of an MHC associated disease sample; and an antibody probe selected from the group consisting of an antibody as described above, a monoclonal antibody derived therefrom, or a derivative thereof; probing the cDNA or genomic library with the antibody probe; and isolating the synthetic antigenic polypeptide detected thereby.

Either cDNA or genomic libraries may be used. The cDNA or genomic libraries may be assembled into suitable vectors that will enable transcription and the subsequent expression of the clone DNA, either in prokaryotic hosts (e.g. bacteria) or eukaryotic hosts (e.g. mammalian cells). The probes may preferably be selected from:

(i) synthetic oligonucieotide probes based on the amino acid sequence of the antigen identified and purified as described above; (ii) antibodies obtained from serum or plasma produced as described above;

(iii) monoclonal or polyclonal antibodies produced against the antigens identified and purified as described above;

(iv) recombinant or synthetic monoclonal antibodies or polypeptides with specificity for the antigen, e.g. as described by Ward et al 1989, Nature 241. pages 544 - 546.

Accordingly in a further aspect of the present invention there is provided a polypeptide encoded by a nucleotide sequence relevant to an MHC associated disease as hereinbefore described or a sequence substantially homologous therewith, active part, derivative, mutant or fragment thereof. Preferably the polypeptide is isolated from a crude protein sample as hereinbefore described or synthetically produced by recombinant or synthetic methods as described above or known to the skilled addressee.

Preferably, the polypeptide is a recombinant polypeptide expressed by a clone containing a nucleotide sequence of a PERB11 gene family or a TNF- related gene family or sequence substantially homologous therewith, derivatives, mutants or fragments thereof as hereinbefore described. The sequences may be haplotype specific, highly polymoφhic and duplicated within the MHC.

The present invention extends to functionally active parts, mutants, derivatives and analogues of the polypeptide which exhibits the desired activity described herein. The polypeptide employed may be "homologous" to the animal being treated meaning that it has the same origin as the species of animals to be treated or it may "heterologous" to the animal being treated meaning that the species of the animal is different.

Nucleotide sequences according to the present invention have been implicated in a number of MHC associated diseases.

Another aspect of the present invention provides a pharmaceutical composition for the treatment or prevention of MHC associated diseases in an animal, said composition including a polypeptide as hereinbefore described and one or more pharmaceutically acceptable carriers and/or diluents. The polypeptide may be a recombinant polypeptide. Preferably the polypeptide is encoded by a nucleotide sequence of the PERB11 gene family or a TNF-related gene family, as hereinbefore described.

For use as a cytotoxic agent in treatment and prevention of MHC associated diseases, the polypeptide may be conjugated to an appropriate cytotoxic agent such as a medicament to treat the MHC disease.

In an even further aspect of the present invention there is provided a pharmaceutical composition containing an antibody as hereinbefore described reactive to a peptide encoded by a nucleotide sequence relevant to an MHC associated disease as hereinbefore described in one or more pharmaceutically acceptable carriers and/or diluents. Preferably the peptide is encoded by a nucleotide sequence of the PERB11 gene family or of a TNF-related gene family, as hereinbefore described.

For use as a cytotoxic agent in treatment and prevention of MHC associated diseases, the antibody may be conjugated to an appropriate cytotoxic agent such as a medicament to treat the MHC disease.

As used herein "pharmaceutically acceptable carriers and/or diluents" include any solvents dispersion media, aqueous solution, proteins, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such agents or pharmaceutically active substances is well known in the art. Supplementary active ingredients can also be incoφorated into the composition including adjuvants.

The pharmaceutical compositions may take a number of forms depending on therapeutic or preventative intent. The pharmaceutical composition may be applied topically or transdermally in the form of ointments, aqueous compositions including solutions and suspensions, liposomes, microcapsules, creams, lotions, aerosol sprays or dusting powders. Applications of the pharmaceutical compositions may be to any area of the body. Altematively, the pharmaceutical compositions may be prepared for oral or parenteral administration. In oral administration capsules or tablets may be prepared in which the pharmaceutical compositions may further include stabilizers, excipients, carriers, preservatives or flavours as is common in the pharmaceutical practice. More specifically, the composition may contain an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft gelatin capsules or maybe compressed into tablets or incoφorated directly with food of the diet. The tablets, troches, pills, capsules and the like may also contain binders such a gum gragacanth, acacia, corn starch or gelatin; excipients such dicalcium phosphate; disintegrating agents such as corn starch, potato starch, algenic and the like; lubricants such as magnesium stearate; sweetening agents such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen or cherry flavouring. When the dosage unit form is a capsule, it may contain in addition to materials of the above type a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills or capsules may be coated with shellac sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl propyl parabens as preservative, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non toxic in the amount employed. In addition the active compound may be incorporated into sustained released preparations and formulas. To improve water solubility, the carrier may be for example a cyclodextrin or serum albumin or a vesicle forming surfactant such as one of the polyethylene alkyl ethers.

For parenteral administration, that is intravenous, intramuscular, subcutaneous or intraperitoneal, the pharmaceutical compositions may further include a pharmaceutically acceptable carrier such as a sterile solution containing other solutes for example sufficient saline or glucose to make the solution isotonic. The present invention will now be more fully described with reference to the following examples. It should be understood however that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described. For example the protocols described are purely exemplary and are not exclusive, other protocols may be used to achieve the same effect.

IN THE FIGURES:

Figure 1 shows nucleotide sequence of a member of the PERB11.1 gene family showing regions characteristic of the PERB11 gene family. Genomic analysis of PERB11.1 reveals polymorphism in both intronic and exonic sequences. Stretches of 1120 bp of PERB11.1 genomic sequence from 5 MHC AHs are compared. The sequence of 57.1 is used as a reference and shown at the top. Nucleotide positions correspond to the positions in the 8892 bp sequence. Dots represent the same nucleotide as in the reference sequence (57.1). Asterisks indicate deletions. Standard ambiguity codes are used where: S=C or G,W=A or T,Y=C or T,M=A or C,R=A or G,V=A or C or G,H=A or C or T and N=A or T or C or G. The cDNA sequence was obtained from poly-A RNA derived from skeletal muscle, using JA3 and JB3 as primers. Asterisks within the cDNA sequence represent nucleotides not found in the cDNA i.e., putative PERB11.1 intron. Nucleotides that are boxed represent exonic differences; amino acids are shown adjacent to the polymoφhic codons. Nonsynonymous amino acid changes, which are not conservative, are shown in white characters on a black background. Leu = leucine, Ala = alanine, Thr = Threonine, Cys = Cysteine, Tyr = Tyrosine, Arg = Arginine, Gin = Glutamine, Val = valine, Glu = glutamic, Lys = lysine. Nucleotide differences are distributed along the region and can be found in both intron and exons. Five of the seven nucleotide changes in the exons lead to non-synonymous amino acid substitutions. The boxed areas show specific regions of the nucleotide sequence which encode the peptide from which antibodies were raised. [From: Immunogentics, (1994), 40, 339-351].

Figure 2 shows the putative protein sequence of the PERB11 as deduced from the nucleotide sequence of Figure 1. PERB11.1 is related to MHC class I molecules. The putative protein sequence of PERB11.1 was deduced from the cDNA sequence, using a coding frame not interrupted by non-sense codons. The amino acid sequence of PERB11.1 including predicted amino acids upstream of the cDNA (see text) was compared with known proteins in the Gene peptide data base version 78, using the BlastP program. The top matches were selected for direct comparison. Amino acids identical to those of PERB11.1 are shaded. Blank spaces represent gaps introduced to obtain the best match between the sequences. Residues 1-92 correspond to the α1 domain and residues 93-135 to the amino terminal part of the α.2 domain of MHC class I molecules. Residues conserved in MHC class I molecules from different species (Grossberger et al. 1992) are shown by vertical arrows and their interactions with other parts of the molecule or an associated molecule (β_2m) are listed. All the top matches are with MHC class I molecules or MHC class I-like molecules, such as the Zn-α2- glycoprotein and the MHC class I-like IgG Fc-receptor from mouse and rat. The overall similarity of PERB11.1 to these molecules is approximately 30% and conserved residues range from 66% to 72%. Six residues (boxed) are shared by all the molecules compared. [From Immunogenetics, (1994), 40, 339-351].

Figure 3 shows the derived PERB11.1 amino acid sequences from 5 MHC ancestral haplotypes. The predicted primary structure of the molecule, when compared to MHC Class I, is shown, b's on top of the sequence represent beta- pleated sheet and a's represent the alpha-helix structure. The boxed regions, PERB11 4D, PERB11 5D and PERB11 6D, were selected for peptide synthesis and for preparation of antibodies.

Figure 4 shows reactivities of two PERB11 antibodies to PERB11 peptides. Reactivity of (a) anti-PB11-4D and (b) anti-PB11-5D were tested by ELISA. Figure 5a shows Pattern 1 kidney 140X. Mouse S/N α5D.

Figure 5b shows Pattern 1 kidney glomerulus 420X. Mouse S/N α5D.

Figure 5c shows Pattern 1 kidney tubules 420X. Mouse S/N α5D.

Figure 6a shows Pattern 1 liver portal tract 420X. Mouse S/n α5D.

Figure 6b shows Pattern 1 rat stomach 140X. Mouse S/N α5D.

Figure 6c shows Pattern 1 heart endomysial 140X. Mouse S/N α5D.

Figure 7a shows Pattern 1 heart endomysial 420X. Mouse S/N α5D.

Figure 7b shows Pattern 1 brain 140X. Mouse S/N α5D.

Figure 7c shows Pattern 1 brain 420X. Mouse S/N α5D.

Figure 8a shows Pattern 1 adrenal cortex 140X. Mouse S/N α5D.

Figure 8b shows Pattern 1 salivary gland 140X. Mouse S/N α5D.

Figure 8c shows Pattern 1 thymus 140X. Mouse S/N α5D.

Figure 9a shows Pattern 1 spleen 140X. Mouse S/N α5D.

Figure 9b shows Pattern 1 testis 140X. Mouse S/N α5D.

Figure 9c shows Pattern 1 testis 420X. Mouse S/N α5D.

Figure 10a shows Pattern 1 ovary 140X. Mouse S/N α5D. Figure 10b shows Pattern 1 ovary 420X. Mouse S/N α5D.

Figure 10c shows Pattern 1 small intestine 140X. Mouse S/N α5D.

Figure 11a shows Pattern 1 small intestine 420X. Mouse S/N α5D.

Figure 11b shows Pattern 1 skin 140X. Mouse S/N α5D.

Figure 11c shows Pattern 1 skin 420X. Mouse S/N α5D.

Figure 12a shows Pattern 2 kidney 140X. Mouse S/N α5D.

Figure 12b shows Pattern 2 kidney tubular cytoplasm 420X. Mouse S/N α5D.

Figure 12c shows Pattern 2 liver 140X. Mouse S/N α5D.

Figure 13a shows Pattern 2 liver cytoplasm 420X. Mouse S/N α5D.

Figure 13b shows Pattern 2 oesophagus of rat stomach 21 OX. Mouse S/N α5D.

Figure 14a shows Pattern 2 mouse stomach 70X. Mouse S/N α5D.

Figure 14b shows Pattern 2 heart 140X. Mouse S/N α5D.

Figure 14c shows Pattern 2 heart 420X. Mouse S/N α5D.

Figure 15a shows Pattern 2 adrenal cortex 140X. Mouse S/N α5D. Figure 15b shows Pattern 2 adrenal cortex spotty cytoplasm 420X. Mouse S/N α5D.

Figure 15c shows Pattern 2 salivary gland 140X. Mouse S/N α5D.

Figure 16a shows Pattern 2 salivary gland spotty cytoplasm 420X. Mouse S/N α5D.

Figure 16b shows Pattern 2 testis 140X. Mouse S/N cc5D.

Figure 16c shows Pattern 2 testis 420X spots and rods 420X. Mouse S/N α5D.

Figure 17a shows Pattern 2 testis filamentous rods 420X. Mouse S/N α5D.

Figure 17b shows Pattern 2 ovary 140X. Mouse S/N α5D.

Figure 17c shows Pattern 2 ovary mesothelial cells 420X. Mouse S/N α5D.

Figure 18a shows Pattern 2 ovary follicle cell cytoplasm 420X. Mouse S/N α5D.

Figure 19a shows Pattern 2 small intestine apical 140X. Mouse S/N α5D.

Figure 19b shows Pattern 2 small intestine apical 420X. Mouse S/N α5D.

Figure 19c shows Pattern 2 skin basal epithelium 140X. Mouse S/N α5D.

Figure 20a shows 5D kidney 140X. Rabbit α5D.

Figure 20b shows 5D kidney glomerulus 420X. Rabbit α5D. Figure 20c shows 5D liver 140X. Rabbit α5D.

Figure 21a shows 5D liver cell surface 420X. Rabbit α5D.

Figure 21b shows 5D testis 140X. Rabbit α5D.

Figure 21c shows 5D testis bright dots 420X. Rabbit α5D.

Figure 22a shows 5D testis filamentous rods 420X. Rabbit α5D.

Figure 22b shows 5D large intestine apical 140X. Rabbit α5D.

Figure 22c shows 5D large intestine apical 420X. Rabbit α5D.

Figure 23a shows 4D heart 140X. Rabbit α4D.

Figure 23b shows 4D heart anti-nuclear 420X. Rabbit α4D.

Figure 23c shows 4D testis 140X. Rabbit α4D.

Figure 24a shows 4D testis bright dots 420X. Rabbit α4D.

Figure 24b shows 4D testis spotty cytoplasm 420X. Rabbit α4D.

Figure 25 shows the nucleotide sequence of the MHC region between HLA

B and the TNF loci.

Figure 26 shows an nucleotide sequence of a substantially homologous TNF-related gene family sequence.

Figure 27 shows a nucleotide sequence of a TNF related gene family sequence without specifying the introns and therefore ignoring those introns containing apparent stop codons. Introns can be deduced by comparison with other TNF-related gene family genes (see Fig. 28 is but one example) and identifying likely 5' and 3' splice sites (see 394-577 which includes a putative intron).

Figure 28 shows a specific example of an amino acid sequence homologous to TNF designated as CL 6.5.40.

Figure 29 shows that two populations of clones from the CL region were isolated from λ libraries of different AHs. The maps represent overlapping clones from the CL region of 7.1 , 57.1 , 8.1 , and 18.2 AHs. Two populations of clones were isolated from three AHs (7.1 , 57.1 , and 8.1). The fragment sizes from the clones correspond to those seen after digestion of genomic DNA. The corresponding clones from 18.2-CL2 have not yet been isolated. Variations in the sizes of Bam HI fragments were observed when the clones from 8.1 and 18.2 were compared with those from 57.1 and 7.1.

EXAMPLE 1 ANTIBODIES TO THE PERB11 GENE FAMILY The PERB11 gene family belongs to the immunoglobulin supergene family and is closely related to MHC Class 1 , neonatal IgG Fc receptor (FcRn) and Zn- alpha-2 giycoprotein all of which appear to be peptide/protein binding receptors. Recent studies have shown that it is a polymorphic gene family with multiple copies within the MHC.

Peptides to PERB11 have been produced that can be used for further characterisation of the gene product and for typing puφoses.

Based upon the amino acid sequence predicted from the PERB11 cDNA sequence (see Figure 1), we have chosen three peptides. These peptides are located in the alpha-1 and alpha-2 domains of PERB11 (figure 2) and were used to immunise rabbits intramuscularly. Rabbits were bled prior to inoculation, and at 4 and 9 weeks post inoculation. The specificity of the antisera was confirmed by testing each serum against all inoculating peptides using an enzyme-linked immunosorbent assay (ELISA), (figure 4).

Specific antibodies to the PERB11 peptides. Two PERB11 peptide antibodies raised in rabbits were tested against three synthesised peptides based on the derived amino acids of PERB11.1 (PB11-4D, PB11-5D and PB11-6D; see Fig. 1). The antisera were also tested against empty wells as a control for nonspecific binding of antisera to the plate. The indirect ELISA system was used to test these antisera.

ELISA plates were coated with streptavidin and then bound to biotinylated peptides. Alkaline phosphatase conjugated sheep anti-rabbit antibody was used as a second antibody to detect the rabbit antisera binding to the plate. Para- nitrophenyl phosphate was used as a substrate for the enzyme and the product was measured at 405nm. The reactivities of "anti-PB11-4D" and "anti-PB11- 5D" to various peptides are shown in (a) and (b) of Fig. 4, respectively. Each curve represents the reactivity of antibody against the peptide specified. X axis represents the time of sera collection and Y axis represents the absorbance at 405 nm. It can be seen that the antisera demonstrate specific activity to the inoculated peptides.

EXAMPLE 2 SPECIFIC REACTIVITY OF A PERB11 ANTIBODY The antibodies produced in Example 1 were tested for their staining patterns in various cells in particular polymorphonuclear neutrophil granulocytes (PMN) and glomerular endothelial cells.

The purpose of this study was to identify membrane proteins of PMNs, and/or glomerular cells, as additional autoantigenic ANCA targets and to characterize the antibodies with respect to their staining patterns. Necrotizing and crescentic glomerulonephristis (NCGN) is frequently associated with circulating antineutrophil cytoplasmic autoantibodies (ANCA). It is established that ANCA are specific for soluble enzymes of granules of polymorphonuclear neutrophil granulocytes (PMN), such as in myeloperoxidase (MPO) in proteinase III (PR3) but scrambled.

Using the antibodies as probes for membrane proteins it was found that antibody PB11 5D was particularly localized in the surface membranes of endothelial cells of human glomerular and renal interstitial capillaries in an ANCA assay.

Such a staining pattern is seen in rapid progressive glomerulonephritis and it is deduced that sequences encoding peptides of PERB11 are found or the cell surface of renal microvascular endothelial cells and are autoantigenic targets for ANCA in patients with active NCGN.

EXAMPLE 3 PROTOCOL FOR STUDYING PERB11 POLYMORPHISM

PERB11 fragments of 2250 bp including the α1 , α2, and α3 domains are amplified by the polymerase chain reaction (PCR) from genomic DNA (Fig. 1). The oligonulceotides selected for PCR are derived from the 5' end of the α1 exon (5' GAGCCCCACAGTCTTCGT-3') and the transmembrane sequence (3'- TGTAAGGTACAAAGACGAC-5') of PERB11 and have been tested using cosmid M32A DNA as a template. With this primer pair no fragment is amplified from cosmid R9A DNA encoding PERB11. PCR is carried out using the GeneAmp reagents and AmpliTaq DNA polymerase (Perkin Elmer Cetus), 0.5μg template DNA and 25pM of each primer. Samples are subjected to 40 cycles of PCR, with intervals at 94°C (1 min), 55°C (1 min) and 72°C (2 min) and the products are directly cloned into the TA vector pCR11 (Invitrogen). Sequences are obtained from double-standed templates using flanking SP6 and T7 primers (Promega) and several oligonucleotides derived from PERB11 intron sequences. These are 42 bp 3' of the α1 exon (3'-GTCTTTTCAATCCCGTC-5^*), 39 and 40 bp 5" and 3" of the α2 exon, respectively (5'-TCACTTGGGTGGAAAGGTGAT-3' and 3'- ACGATCTCAACGGAGTGGAGG-5') and 40 bp 5' of the α3 exon (5'- GTTCCTCTCCCCTCCTTAGA-3'). All of the PERB11 alleles are verified by sequencing of at least 3 clones derived from 2 independent PCT products.

Primers JPIA! (5' AGG ACA CGA TGT GCC AAC AG 3') and JPIB2 (5' CTT CCA CCA CCT TCA TGC TC 3') were used to amplify a genomic fragment of 1270 bp from five B lymphoblastoid cell lines, each homozgous for an ancestral haplotype [(Q6-3975:7.1=HLA-A3, -Cw7,-B7,TNFL BAT3LC2C, BF S, C4A3, C4B1 , HLA-DR2-DQ6;R6-12337;57.1 = HLA-a1 , -Cw6, -B57, TNFLBAT3 L C2 C, BF S, C4A6, C4B1 , HLA-DR7- DQ9; R6-12373; 8.1= HLA-A1 , - Cw7, -B8, TNFSBAT3SC2C, BFS, C4AQ0, C4B1 , HLA-DR3, DQ2; R5-5054: 18.2 = HLA- A30, CW5, B18, TNFLBAT3 S, C2 C- BF-F1 , C4, A3, C4BQ0, HLA-DR3, -DQ2 and R6-12361 : 46.1 = HLA -A2, Cw1 , -B46, TNFL BAT3 L C2 C BF S, C4A4, C4B2, HLA-DR9, -DQ9] (Wu et al 1992, Hum. Immunol, 33. 89-97; Degli-Esposti et al 1993, Hum. Immunol, 38, 3-16). 250 ng of DNA extracted from these cell lines was used for PCR amplification. Each reaction contained 200 μm each of dATP. dCTP, dGTP, and dTTp 2 mM Tris-HCl (ph) 8.3). 3mM magnesium chloride 50 mM KCI 50 pmol of the primers and 1 unit of Taq DNA polymerase (Amplitaq: Cetus, Emeryville, CA)in a total volume of 50 μl. The reaction was overlaid with light mineral oil Sigma, St Louis MO) and subjected to 95°C for 2 min then thermocycling (35 cycles of 95°C for 45 s 55°C for 45 s, 72°C for 2 min) followed by a final extension at 72°C for 10 min [(Gene Amplimer; Perkin Elmer (Norwalk, CT) and Cetus)]. The 1270 bp products obtained were fractionated through 1% low melting temperature (Seaplaque: FMC Bioproducts, Rockland ME) and further purified by Centricon column centrifugation (Centricon-100 microconcentrators Amicon, Division of W.R. Grace & Co., Danvers, MA) according to the manufacturer's directions. Both stands of the products were sequenced, using JP1A1 , JP1B2, and five intemal primers: JA3 (5' GTT CAT GGC CAA GGT CTG AG 3'), JP1A2 (5^* TGG GCT GAG TTC CTC ACT TG 3"), JP1B1 (5' GGT CCT TGA TAT GAG CCA GG 3'), JB3 (5' GTC AGG GTT TCT TGC TGA GG 3'), and JB4 (5' ACA GAT CCA TCC CAG GAC AG 3') in a fluorescent-labeled dideoxy termination reactions analyzed on an automated 373A DNA sequencer (Applied Biosystems).

EXAMPLE 4

ANTIBODIES TO PERB11 PEPTIDES - REPORT OF PATTERNS SEEN

BY INDIRECT IMMUNOFLUORESCENCE ON RODENT SUBSTRATE OF

ANTI-RABBIT SERA AND ANTI-MOUSE MONOCLONAL SUPERNATANTS

Materials and Methods

Peptide Syntheses

Two systems were used to produce the PERB11 peptides. The multiple antigen peptide (MAP) system developed by Posnett et al (J Biol Chem 263:1719-1725, 1988) were used to produce PB11-1M, 2M and 3M, kindly provided by Dr Posnett, the Basel Institute for Immunology, Basel, Switzerland. Each peptide antigen consists of a branching lysine core with 8 copies of the pepitde linked to the core by the COOH-terminal of the amino acid. The complex has a high immunogenicity without the need to conjugate to a protein carrier and yields a good response to the linked peptide in animals injected (J Biol Chem 263:1719-1725, 1988). The other three peptides, PB11-4D, 5D and 6D (purchased from the Chiron Mimotopes Pty Ltd, Clayton, Victoria 3168, Australia) were synthesised and conjugated to the diphtheria toxoid. The six peptide were used to immunise rabbits as described below.

Immunisation Procedure

For PB11-4D, 5D and 6D, 1 mg of each peptide was dissolved in 400 ul of sterile distilled water. 400 ul of each peptide solution was added to 400 ul of Titremax adjuvant and emulsified by sonification for 90 seconds on ice. 40 ul of each emulsion was injected intramuscularly into each hind leg of rabbits. Rabbit sera were collected between 9 and 14 weeks of post immunisation. One mg of each PERB11-1M, 2M and 3M peptides was used to make 50% emulsions with complete freund's adjuvant and injected subcutaneously into 3 rabbits with one peptide per one rabbit. The same emulsions were prepared and injected weekly for another 3 weeks before the collection of rabbit sera on the fourth week. All sear were tested for activities against the corresponding peptides using the indirect ELISA technique.

ImmunopreciDitation

Indirect Immunofluorescence of Various Tissues

Rats, mice and guinea pigs were obtained from the Animal House of the Research Centre at Royal Perth Hospital. The animals were dissected, and tissues were combined into composite blocks and were frozen in liquid nitrogen within 1 hour of animal death using Tissue Tek OCT embedding medium (Miles Inc, USA). The tissues used were rat stomach, kidney, liver, heart, ovary, testis, skin (tail), small intestine, large intestine, brain, adrenal, thymus, spleen and salivary gland, together with mouse stomach and guinea skeletal muscle.

Cryosections of 4μm thickness were air dried and covered with sera from rabbits immunised with PB11-2M, 4D and 5D. After incubation, the sections were washed, then stained with FITC conjugated sheep anti rabbit Ig, F(ab')₂ fragment, affinity purified (Silenus Laboratories, Australia). The sera were diluted 1 :10 and the conjugate 1 :40 in PBS PH7.6. After the second incubation, slides were washed 3 times then mounted in glycerol based semi-permanent medium pH 9.1 (Immunoconcepts, USA) and read blind by at least two independent readers on an immunofluorescence microscope. A conjugate control section (FITC sheep anti rabbit Ig only) was included on each run.

In addition, the rabbit sera were tested on ethanol fixed human neutrophils. Neutrophils from a normal human donor (Blood group O, positive) were sprayed onto glass slides using cytocentrifuge (Shandon Elliott). The slides were air dried and then fixed in cold ethanol for 10 minutes. The rabbit sera were tested by indirect immunofluorescence and read as described above, except the FITC conjugate was diluted at 1:800. Results

PATTERN 1

Routine Composite Block

Kidney

There is a continuous outline of the renal tubules in addition to reticulin-like fibres in the spaces between the tubules. The glomerulus fluoresces in a similar fashion with a continuous outline of Bowman's capsule and strands inside the glomerulus which appear to outline circular structures, possibly capillaries (Figs.

5a, b and c).

Liver

There is very weak fluorescence in reticulin-like pattern between the hepatocytes of the parenchyma. Thin connective tissue outlining the portal tracts fluoresces more brightly (Fig. 6a).

Rat Stomach

There is a reticulin-like fluorescence in between the cells and glands of the gastric mucosa. There is a weaker fluorescence in between the smooth muscle cells of the muscularis externa and muscularis mucosa (Fig. 6b).

Mouse Stomach

Fluorescence in the smooth muscle layers is similar to that in the rat stomach. There is no fluorescence in the mucosa (see Fig. 6b).

Heart

Fluorescence occurs in an endomysial pattern between the myocardial cells. Small circular structures are visible as part of this pattern. There is no fluorescence within the myocardial cells (Figs. 6c and 7a) Composite Block 1

Brain

A dark black background is punctuated by scattered wispy strands and ring-like structures (Fig. 7b). At high power a few of the larger ones are clearly blood vessels but most are not cleariy identifiable. They may cells or some form of fibres.

Adrenal Gland There is fluorescence in a network-like pattern occurring between and outlining the cells of the adrenal cortex (Fig. 8a).

Salivary Gland

Fluorescence creates a honeycomb-like network around the acini and ducts and highlighting the division between lobules of the gland (Fig. 8b).

Thvmus

There is fluorescence of connective tissue outlining lobules and surrounding blood vessels. In the substance of the lobules themselves there are wispy strands and ring-like structures, probably representing connective tissue fibres (Fig. 8c).

Spleen

There is a clear contrast between the white pulp, which is mainly dark, and the red pulp in which there is abundant fluorescence. The white pulp has a thin outline, with fluorescence around the central artery and sparse wispy strands in the substance of the lymphoid tissue. In the red pulp, there is abundant fluorescence in between cells (Fig. 9a). Composite Block 2

Testis

There is a fluorescence at the lining of the seminiferous tubules, probably the basement membrane. In the spaces between the tubules, blood vessel walls are highlighted but in addition there are numerous bright, small, patent ring-like structures, and similar wispy longitudinal strands which appear as double lines. These may represent fibres whose surfaces are fluorescing and being viewed in transverse section (rings) and longitudinal section (strands) (Figs. 9b and c).

Ovary

There is fluorescence of the tunica albuginea but not the overlying mesothelial cells. Most of the ovary is composed of follicles in various stages of development, and corpus lutei. Within all these follicles there is reticulin-like fluorescence in between the cells. In those follicles that are cystic, the lining of the cysts is highlighted (Figs 10a and b).

Small and Large Intestines

There is diffuse connective tissue-like fluorescence throughout these tissues, in particular, the endomysium of the smooth muscle and connective tissue stalks of the villi (Figs. 10c and 11a).

Skin

Fluorescence occurs at the basement membrane at the dermo-epidermal junction, hair follicles and sebaceous glands. In addition, there is bright fluorescence of wispy strands and ring-like structures scattered through the dermis, particularly in association with the skin appendages. They are similar to the structures seen in between the seminiferous tubules of the testis (Figs 11b and 11c). Discussion

PATTERN 1

This is a pattern of connective tissue fluorescence. It appears to fluoresce basement membranes (eg seminiferous tubules, skin), reticulin-like connective tissue between cells in parenchymal organs (eg liver, ovary, spleen) and endomysium. Several tissues show a combination of firstly, small, patent, bright ring-like structures and secondly, wispy strands which appear as double lines. This is seen most obviously in the testis and skin but similar structure may be visible in other tissues such as brain and intestine. One hypothesis is that a connective tissue fibre such as collagen may be showing fluorescence of antigens on its surface. When seen in transverse section they would appear as rings and when seen in longitudinal section would appear as lines. Candidates for such a surface antigen may include fibronectin or an integran protein.

PATTERN 2

Routine Composite Block

Kidney

A section of kidney shows dark glomeruli surrounded by tubules whose cell cytoplasm displays moderate fluorescence. On high power, the cytoplasmic fluorescence is granular and spotty rather than smooth. There is no fluorescence in the spaces between the tubules (Figs. 12a and b).

Liver

The liver section appears homogeneous with a smooth fluorescence of the cytoplasm of all hepatocytes, leaving dark nuclei (Figs. 12c and 13a). Rat Stomach

The rat oesophagus (Fig. 13b), which forms part of this section, displays fluorescence of the first 1-3 layers of the basal epithelium, as well as the surface keratin. In the stomach itself, there is strong anti-parietal cell fluorescence in the mucosa, with bright vertical columns of mucosal cells, but not those nearest the surface (mucous cells) or at the base of the glands (chief cells). There is no fluorescence in the other layers of the rat stomach (Fig. 13c).

Mouse Stomach Anti-parietal cell activity is also present, but not as bright as in the rat stomach. In addition, there is fluorescence of the smooth muscle of the muscalaris externa and muscalaris mucosa, with vertical fibres in the mucosa (Fig. 14a).

Heart

There is streaky longitudinal fluorescence in the cytoplasm of the myocardial cells, Endomysial fluorescence is also visible as a fuzzy outline to the cells (Fig. 14b and c).

Composite Block 1

Adrenal Gland

Fluorescence occurs in the cytoplasm of the cells of the adrenal cortex (Fig. 15a). High power reveals that the cytoplasmic fluorescence is distinctly spotty and granular (Fig. 15b).

Salivary Gland

Fluorescence occurs in the cytoplasm of the cells of the tubules and acini (Fig. 15c), and on high power (Fig. 16a), like in the adrenal gland, is spotty and granular. Composite Block 2

Testis

There appears to be spotty cytoplasmic fluorescence of most of the cells at the base of the seminiferous tubules. There is increasing fluorescence towards the centres of the tubules, but difficult to localise to any particular structure or to an exclusively intracellular of extracellular location (Fig. 16b). On high power (Fig. 16c and 17a), there are abundant long, thin, rod-like structures. These filamentous structures occur in parallel clusters of 2-4 and are radially oriented towards the lumen, except for those in the middle of the lumen which are haphazard. They are in the right location to be sperm but are probably too long, thin and straight, and in condition, structures that are more likely to be sperm are visible as separate black shadows. One possibility is that they could represent crystallisations of a protein contained in the fluid in the tubules.

Ovary

There is fluorescence of the surface mesothelial cells but not the underlying tunica albuginea. There is spotty fluorescence in the cytoplasm of cells in the ovarian follicles (Figs. 17b, c and 18a).

Small and Large Intestines

There is bright fluorescence of the cytoplasm of mucosal cells. There is particularly bright at the apical/luminal surface of these cells, creating bright fluorescence facing the lumina of glands (see Figs. 18b and c, 19a and b).

Skin

Fluorescence occurs at the 1-3 basal layers of the epidermis. As invaginations of this layer, the basal cells of the hair follicles and sebaceous glands also fluoresce. In addition, there is staining of keratin at the surface and in hair shafts (Fig. 19c). Discussion

PATTERN 2

Pattern 2 consists of cytoplasmic fluorescence in most tissues, which in many cases (eg kidney, adrenal, salivary gland, ovary) is spotty and granular. It shows a resemblance to anti parietal cell, anti mitochondrial and anti liver and kidney microsomal antigens, but is neither of these. The best summary that can be given at this stage is of a non-tissue specific cytoplasmic antigen.

ANTI-5D PATTERNS

Kidney

A spotty pattern is present in every glomerulus. It consists of specks, approximately 10 micrometres in diameter, occurring towards the periphery of the glomerulus, and 7-10 per glomerulus in number. Some give the impression of being crescent shaped, suggesting that they may be endothelial cells. However there is no such fluorescence of endothelial cells elsewhere in the kidney, either small vessels in the interstitium or the endothelial cells of larger vessels (whose walls do fluoresce in pre-immune and post-immune sera). If they are endothelial cells then this suggests some antigen which is specific to endothelial cells in the glomerulus. Other alternatives are mesangial cells (which would be expected to be more centrally located) or epithelial cells (Fig. 20a and b).

Liver The pre-immune serum shows fluorescence of the cytoplasm of hepatocytes with highlighting of the cell membrane. This pattern becomes brighter in the post-immune serum (Figs. 20c and 21a).

Rat Stomach The pre-immune serum shows fluorescence of the smooth muscle of the wall and weakly in the mucosa. In the post-immune, the smooth muscle activity is much brighter. In addition, there is new fluorescence in the cytoplasm of mucosal cells with highlighting of the luminal surface of glands at the base of the gastric mucosa.

Testis The pre-immune serum produces weak fluorescence of the basement membrane of the seminiferous tubules, with sparse fine dots in the lumen. In the pre-immune serum, there is the addition of bright fluorescence of the wall of blood vessels, plus a mixture of numerous fine bright dots, apparently associated with the surface cells in the tubules, and fine filamentous rods in the lumen (Figs. 21b, c and 22a).

Skin

The basal epithelium of the epidermis and hair follicles fluoresces more brightly in the post-immune compared to the pre-immune.

Small and Large Intestines

There is an increase in smooth muscle activity as compared to the pre- immune. In addition, there is cytoplasmic fluorescence in the mucosa which highlights the luminal/apical surface of glands in a similar fashion to supernatant patter 2 (Figs. 22b and c).

ANTI-4D PATTERNS

Heart The pre-immune serum shows cytoplasmic fluorescence with an accentuation of the intercalated discs. The post-immune serum shows the addition of bright, fine dots in the cytoplasm of myocardial cells, which are approximately one per cell and centrally located (Figs. 23a and b). Testis

The pre-immune picture is identical to that in the pre-immune 5D; that is, weak fluorescence of the basement membrane of tubules, blood vessels and sparse fine dots in the lumina of the seminiferous tubules. The post-immune serum, however, produces a spectacular pattem in which the lumina of the tubules contain a ring of abundant, fine dots, apparently associated with the surface cells of the tubule. These are also seen in the rabbit anti-5D serum but not nearly as brightly. By their position they could be sperm heads but there are separate structures in the tubules, appearing as a negative black outline, which appear to be sperm. An additional feature in the anti-4D is that some of the cells at the basal aspect of the tubules, adjacent to the basement membrane, show spotty cytoplasmic fluorescence. This applies for approximately one in three cells at the basal layer (Figs. 23c, 24a and b).

SIMILARITIES BETWEEN RABBIT AND SUPERNATANT PATTERNS

The immunofluorescence patterns produced by the rabbit sera and the mouse supernatants share common findings in the intestine, testis and basal epithelium in the skin and oesophagus.

Small and Large Intestines

Fluorescence of the apical surface of mucosal cells in the intestine is common to the rabbit anti-5D serum and supernatant pattem 2. It produces a bright highlighting of the luminal surface of glands in the mucosa. This is brighter and more intense in the supernatant, which has a dark background, compared to the rabbit serum in which there is also anti-smooth muscle activity and nonspecific background staining. The rabbit anti-5D also produces some apical fluorescence of glands at the base of the gastric mucosa in the rat stomach. Testis

Fluorescence of the basement membrane is a feature of supernatant pattem 1. This is also present in the rabbit anti-4D and anti-5D but no different from the pre-immune. Abundant bright fine dots are present in the lumina of the tubules in the anti-4D and to a lesser extent in the anti-5D. In addition, the rabbit anti-5D tubule contains straight, fine, radially oriented filamentous rods shared with supernatant pattem 2. These rods could represent a crystallation of proteins contained in the tubular fluid. Additional features in the testis are fluorescence of blood vessel walls in the anti-5D and supernatant pattem 1 , and the wispy strands and ring-like structures, which could represent some form of connective tissue fibre, between the seminiferous tubules in supernatant pattern 1.

Basal Epithelium

The basal epithelium of the oesophagus of the rat stomach and skin fluoresces in supernatant pattens 2 and 3. A similar patter occurs in the skin in the rabbi anti-5D; the oesophagus in the anti-5D is equivocal because of the high level of background activity in this tissue. Basal epithelial activity is not a feature of supernatant pattern 1. The basal epithelial pattem takes the form of fluorescence in the cytoplasm of the cells in the 1-3 basal layers of the epithelium. In the skin, this includes activity at the basal cells lining the hair follicles and sebaceous glands, which are invaginations of the basal layer of the epidermis.

By reference to these features it is possible to define an antibody to the PERB11 gene products.

EXAMPLE 5

GENOMIC ORGANIZATION OF A POLYMORPHIC DUPLICATED

REGION OF CENTROMERIC OF HLA-B

The region between tumor necrosis factor (TNF) and HLA-B in the central major histocompatibility complex (MHC) is polymoφhic and associated with several autoimmune diseases. The polymoφhisms are haplospecific or haplotypic and retained within the same MHC ancestral haplotype (AH). We have cloned this region from four AHs into λ bacteriophage and found that a highly polymoφhic region in the TNF-HLA-B inverval is duplicated. Clones from this region isolated from three MHC AHs show two populations.

Materials and Methods Isolation of λ genomic clones. Well defined homozygous cell lines, Q6/3975 (HLA A3, Cw7, B7, C2C, BfS, C4A3, C4B1 , DR2, Dqw6) and R5/5054 (HLA A30, Cw5, B18, C2C, BfF1 , C4A3, C4BQ0, DR3, Dqw2), carrying the 7.1 and 18.2 AHs respectively, were used to construct the λ DNA libraries as described previously (Abraham et al.1991 , Immunogenetics 33, 50-33). The EMBL3 and lambdaGEM-11 bacteriophages were used as cloning vectors for the 18.2 and 7.1 AHs respectively. The two DNA libraries and the previously established 57.1 and 8.1 libraries (Abraham et al, 1991) were used to isolate clones in the TNF to HLA-B region, including the polymoφhic region by using ³²P- labeled genomic walking probes (Abraham et al, 1991 ; Spies et al. 1989a; Chimini et al. 1990, Science, 243. 214-217). Library screening and purification of clones were performed according to standard methods (Maniatis et al. 1982, see Ref. A).

Restriction mapping, λ DNA was digested with Bam HI, Bam Hl+Sal I, Xba I and Xbal+Sal I (Promega, Madison, Wl) according to the manufacturer's specifications. The digested DNA samples were electophoresed through an 0.8% agarose gel (LE, Dekem) at 1.3 V/cm for 18h. The DNA digests were observed under ultraviolet (UV) light after staining with ethidium bromide. DNA was transferred to membranes (Gene Screen Plus; Dupont, Wilmington, DE) and hybridized with specific genomic probes derived from the polymoφhic region of interest (Wu et al. 1992, Human Immunol. In press). The methods of Southern blotting and hybridization were as previously described (Dawkins et al. 1989, Hum. Immunol, 26: 91-97). Selected clones were used to map the Bam HI sites by partial digestion with the enzyme and probing with oligonucleotides specific for the left and right arms of the λ vector (Lambda Map System; Promega). All hybridization membranes that were used for reprobing were stripped and carefully checked for the absence of signal as previously described (Tokunaga et al. 1988; Zhang et al. 1990).

Pulsed field gel eletrophoresis (PFGe) analysis. Genomic DNA plugs of homozygous cell lines, prepared as previously described (Tokunaga et al. 1988, J. Exp. Med., 168. 933-960) and carrying 57.1 , 8.1 , 18.2 and 7.1 were digested with Bss HII, Sac II, Cla I, and Sac ll+CIa I. For Bss HN, the digested DNA were run on 1.5% agarose gel at 15°C, 150 V with 35 s switching time for 20 h. The switching time was changed to 25 s when running Sac II, Cla I and Sac ll+CIa I digested DNA in order to separate the smaller fragment. Using these running conditions fragments of 2.0 kilobase (kb) and above were retained in the gel. Blotting and hybridization were as previously described (Tokunaga et al. 1988; Zhang et al. 1990, J. Exp. Med., 171. 2101-2114). A TNFA probe was used as described previously (Dawkins et al. 1989; Pennica et al. 1984, Nature, 312. 724-729).

Results The CL region is duplicated and polymorphic. A previous restriction fragment length polymoφhism (RFLP) study (Wu et al. 1992) indicates that a region between BATI and HLA-B is extremely polymoφhic using a cloned genomic probe located approximately 35 kb centromeric of HLA-B (Spies et al. 1989a). In all AHs tested, two hybridizing fragments were detected. The presence of two bands in the RFLP study prompted the current study to determine whether, in fact, two related loci were present, λ genomic clones of the region were isolated from four AHs; 8.1 , 18.2, 57.1 , and 7.1. Results from 8.1 , 57.1, and 7.1 showed that two populations of overlapping clones (Fig. 5) were present. The two cloned regions, designated CL1 and CL2 were identified as carrying a hybridizable 6.5 kb or 4.5 kb Bam HI fragment, respectively. Although clones representing the CL2 region of 18.2 have not been isolated, the results indicate that a second locus is also present in this haplotype (see below). The results were confirmed by Southern hybridization analysis of genomic DNA. After digestion with Bam HI, two fragments (6 kb and 4.5 kb) of equal intensity were observed in 8.1 , 57.1, 7.1, and 18.2. The Southern analysis confirmed that the hybridizing regions were duplicated in all four AHs and marked by two Bam HI fragments of approximately 6 kb and 4.5 kb.

Discussion As part of a general approach to the study of genes within the central MHC, we have developed a strategy to identify loci that are important with respect to immune function. Our approach relies upon the fact that many, if not all, immunologically relevant genes will be polymorphic and specific alleles will be carried as part of a particular AH. As described for many loci within the MHC, polymoφhism is often a manifestation of duplicated loci. As part of our current studies we have initiated an assessment of whether mapping the regions of duplication is useful in identifying critical genomic sequences involved in immunological functions.

We have chosen a region in the central MHC that remains devoid of characterized transcriptionai units, but within which, at least candidate autoimmune disease susceptibility genes may be present (Degli-Esposti et al. 1992). Previously, we have established that the region of interest is extremely polymoφhic and is due, in part, to sequence variation in duplicated, reiterated sequence elements called "haplospecific geometric elements" (Abraham et al. 1992). We have now extended the characterization of the region containing these elements and have demonstrated that the geometric elements form part of duplicated units, designated CL1 and CL2, that are polymoφhic when comparisons between AHs are made. Both loci are within the central MHC, directly duplicated and within approximately 16 kb of each other. This arrangement is reminiscent of other regions in the MHC where duplicated genes are encoded. If our hypothesis is correct then the CL region will contain genes that are significant in terms of immunoregulation. Finally it is to be understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as outline herein.

Claims

CLAIMS:

1. A nucleotide sequence coding for a gene relevant to an MHC associated disease, or gene sequence substantially homologous therewith, derivative, mutant or fragment thereof, said sequences being haplotype specific, highly polymoφhic and duplicated within the MHC.

2. A nucleotide sequence according to claim 1 wherein said sequence is haplotype specific, highly polymoφhic and duplicated within a distinct gene family of the MHC.

3. A nucleotide sequence according to claim 2 wherein the gene family is a PERB11 gene family.

4. A nucleotide sequence according to any one of claims 1 to 3 having a nucleotide as illustrated in Figure 1 or sequence substantially homologous therewith, active part, derivative, mutant, analogue or fragment thereof.

5. A nucleotide sequence according to any one of claims 1 to 4 wherein the nucleotide sequence is from nucleotide 7686 to 7944 or nucleotide 8218 to 8359 as illustrated in Figure 1 or sequence substantially homologous therewith, active part, derivative, mutant, analogue or fragment thereof.

6. A nucleotide sequence according to any one of claims 1 to 5 having a nucleotide sequence from nucleotide 7751 to 7944 or nucleotide 8218 to 8343 as illustrated in Figure 1 or sequence substantially homologous therewith, active part, derivative, mutant or fragment thereof.

7. A nucleotide sequence according to any one of claims 1 to 6 having a nucleotide sequence from nucleotide 7793 to 7825, or nucleotide 7857 to 7889 or nucleotide 8254 to 8298 as illustrated in Figure 1 or sequence substantially homologous therewith, active part, derivative, mutant or fragment thereof.

8. A nucleotide sequence according to claim 2 wherein the gene family is a TNF-related gene family.

9. A nucleotide sequence according to any one of claims 1 , 2 or 8 having the sequence substantially as illustrated in Figure 25 or sequence substantially homologous therewith, fragment, derivative, mutant and/or analogue thereof.

10. A nucleotide sequence according claim 8 or 9 having a homologous nucleotide sequence corresponding to the sequence in Figure 26 or sequence substantially homologous therewith, fragment, derivative, mutant and/or analogue thereof.

11. A nucleotide sequence according to any one of claims 8, 9 or 10 having a nucleotide sequence corresponding to the sequence from nucleotide 341 to 367, nucleotide 383 to 394 or nucleotide 578 to 610 substantially as illustrated in Figure 27 or a sequence substantially homologous therewith, active part, derivative, mutant, fragment and/or analogue thereof.

12. An amino acid sequence encoded by a nucleotide sequence according to claim 1 or 2.

13. An amino acid sequence of a PERB11 gene family encoded by a nucleotide sequence as illustrated in Figure 1 or substantially homologous sequence, active part, derivative, mutant or fragment thereof.

14. An amino acid sequence according to claim 13 wherein the nucleotide sequence is from nucleotide 7687 to 7944 or from nucleotide 8218 to 8359 as illustrated in Figure 1 or substantially homologous sequence, active part, derivative, mutant or fragment thereof.

15. An amino acid sequence according to claim 13 or 14 encoded by a nucleotide sequence from nucleotide 7751 to 7944 or nucleotide 8218 to 8343 as illustrated in Figure 1 or a sequence substantially homologous, active part, derivative, mutant or fragment thereof.

16. An amino acid sequence according to any one of claims 13 to 15 encoded by a nucleotide sequence from nucleotide 7793 to 7825 or nucleotide 7857 to 7889 or nucleotide 8254 to 8298 as illustrated in Figure 1 or sequence substantially homologous therewith, active part, derivative, mutant or fragment thereof.

17. An amino acid sequence according to any one of claims 13 to 16 wherein the amino acid sequence is YDRQKCRAKPQ, KTWDRETROLT or IHEDNSTRSSQHFYY or a sequence substantially homologous therewith, active part, derivative, mutant or fragment thereof.

18. An amino acid sequence of a TNF-related gene family encoded by a nucleotide sequence of Figure 25 or sequence substantially homologous therewith, derivative, mutant or fragment thereof.

19. An amino acid sequence of a TNF-related gene family encoded by the nucleotide sequence of Figure 26 or sequence substantially homologous therewith, derivative, mutant or fragment thereof.

20. An amino acid sequence according to claim 18 or 19 encoded by a nucleotide sequence corresponding to the sequence from nucleotide 341 to 367, nucleotide 383 to 394 or nucleotide 578 to 610 substantially as illustrated in Figure 27 or a substantially homologous sequence, active part, derivative, mutant, fragment and/or analogue thereof.

21. An amino acid sequence according to any one of claims 18 to 20 encoded by a nucleotide sequence as illustrated in Figure 28 and designated CL.6.5.40.

22. An amino acid sequence according to any one of claims 18 to 21 encoded by an amino acid sequence NLAHLVGDAL, WQWL or GIYSLKGKCSF respectively or a sequence substantially homologous therewith, active part, derivative, mutant or fragment thereof.

23. A method of preparing a cDNA sequence of a gene relevant to an MHC associated disease said method including the steps of: providing a source of animal cells; isolating the mRNA from the cells; treating the mRNA to produce cDNA; and probing for cDNA of genes identified as relevant to the aetiology of an MHC associated disease, said cDNA sequence being haplotype specific polymoφhic and duplicated within a distinct gene family of the MHC.

24. A method according to claim 23 wherein the gene family is PERB11 gene family or a TNF-related gene family.

25. A DNA clone which expresses a nucleotide sequence of a gene relevant to a MHC associated disease, said gene being haplotype specific, highly polymoφhic and duplicated within the MHC.

26. A DNA clone according to claim 25 wherein the nucleotide sequence is from a distinct gene family of the MHC.

27. A DNA clone according to claim 26 wherein the gene family is a PERB11 gene family and the nucleotide sequence is according to any one of claims 4 to 7.

28. A DNA clone according to claim 26 wherein the gene family is a TNF- related gene family and the nucleotide sequence is according to any one of claims 9 to 11.

29. A recombinant polypeptide encoded by a DNA clone according to any one of claims 25 to 28.

30. An antibody reactive to a polypeptide or native protein relevant to an MHC associated disease, said polypeptide or native protein encoded by a nucleotide sequence being haplotype specific, highly polymorphic and duplicated within the MHC.

31. An antibody according to claim 30 wherein the nucleotide sequence is within a distinct gene family of the MHC.

32. An antibody according to claim 31 wherein the gene family is a PERB11 gene family and the polypeptide has an amino acid sequence according to any one of claims 13 to 17.

33. An antibody to a PERB11 gene family which is PB11-4D, PB11-5D or PB11-6D as hereinbefore described or an antibody having similar specificity.

34. An antibody according to claim 31 wherein the gene family is a TNF- related gene family and the polypeptide has an amino acid sequence according to any one of claims 18 to 22.

35. A method of identifying a protein or polypeptide related to an MHC associated disease including obtaining genomic DNA from an MHC associated disease sample; translating a nucleotide sequence from the genomic DNA coding for a gene relevant to an MHC associated disease or gene sequence substantially homologous therewith, derivative, mutant or fragment thereof, said sequence being haplotype specific highly polymoφhic and duplicated in the MHC; obtaining a polypeptide from the translated nucleotide sequence; raising an antibody to the polypeptide or native protein which antibody is produced by a method including challenging an animal with the polypeptide or native protein; harvesting antibodies produced from said animal in the serum or plasma, or from immune cells of the animal; probing an MHC associated or non-MHC associated disease sample with the antibody; comparing patterns of an MHC associated and non-MHC associated disease samples obtained by probing to identify a protein related to an MHC associated disease.

36. A method according to claim 35 wherein the nucleotide sequence is derived from a distinct gene family of the MHC.

37. A method according to claim 36 wherein the gene family is PERB11.

38. A method according to claim 37 wherein the nucleotide sequence is according to any one of claims 4 to 7.

39. A method according to claim 36 wherein the gene family is a TNF-related gene family.

40. A method according to claim 39 wherein the nucleotide sequence is according to any one of claims 9 to 11.

41. A method of purifying an MHC associated disease polypeptide, said polypeptide encoded by a nucleotide sequence which is haplotype specific, highly polymorphic and duplicated within a distinct gene family of the MHC which method includes providing: a crude protein mixture; an antibody against a polypeptide encoded by a nucleotide sequence relevant to an MHC associated disease said nucleotide sequence being haplotype specific, highly polymoφhic and duplicated with a distinct gene family and immobilized on a suitable support, which antibody is produced by a method including challenging an animal with a peptide encoded by a nucleotide sequence relevant to an MHC associated disease as hereinbefore described; harvesting antibodies produced from said animal in the serum or plasma; subjecting the protein mixture to an affinity chromatography utilizing the immobilized antibody; and isolating the purified polypeptide.

42. A method according to claim 41 wherein said gene family is a PERB11 gene family and the antibody is according to any one of claims 32 or 33.

43. A method according to claim 41 wherein said gene family is a TNF-related gene family and the antibody is according to claim 34.

44. A method for treating or preventing an MHC associated disease in an animal, said method including administering to said animal an effective amount of a polypeptide derived from a nucleotide sequence relevant to an MHC associated disease or sequence substantially homologous therewith, derivatives, mutants or fragments thereof, said sequences being haplotype specific, highly polymoφhic and duplicated within the MHC.

45. A method according to claim 44 wherein the nucleotide sequence is derived from a distinct gene family of the MHC.

46. A method according to claim 45 wherein the gene family is a PERB11 gene family or a TNF-related gene family.

47. A method according to claims 44, 45 or 46 wherein the MHC associated disease is selected from the group including Psoriasis, Nasopharyngeal carcinomas and Spondyloarthopathies, Myasthenia gravis, IgAd and CVI, cerebral malaria, rheumatoid arthritis, AIDS and cachexia.

48. A method for diagnosing an MHC associated disease in an animal, said method including: providing a sample from a patient suspected of having an MHC associated disease; providing a probe related to an MHC associated disease said probe indicative of a nucleotide sequence which is haplotype specific highly polymoφhic and duplicated within the MHC; probing the sample with the probe; and comparing the sample with a sample derived from a patient which does not have the MHC associated disease.

49. A method according to claim 48 wherein the nucleotide sequence is derived from a distinct gene family of the MHC.

50. A method according to claim 49 wherein the gene family is a PERB11 or a TNF-related gene family.

51. A method according to claims 48 to 50 wherein the MHC associated disease is selected from the group including Psoriasis, Nasopharyngeal carcinomas and Spondyloarthopathies, Myasthenia gravis, IgAd and CVI, cerebral malaria, rheumatoid arthritis, AIDS, cachexia.

52. A method for preparing a synthetic antigenic polypeptide relevant to an MHC associated disease which method includes providing a cDNA library, or genomic library derived from a sample of an MHC associated disease sample; and an antibody probe selected from the group consisting of an antibody according to any one of claims 30 to 34, a monoclonal antibody derived therefrom, or a derivative thereof; probing the cDNA or genomic library with the antibody probe; and isolating the synthetic antigenic polypeptide detected thereby.

53. A synthetic antigen polypeptide prepared according to claim 52.

54. A pharmaceutical composition for the treatment or prevention of an MHC associated disease in an animal, said composition including a polypeptide prepared by a method according to any one of claims 41 to 43 and one or more pharmaceutically acceptable carriers and/or diluents.

55. A pharmaceutical composition containing an antibody according to any one of claims 30 to 34 reactive to a peptide or native protein encoded by a nucleotide sequence relevant to an MHC associated disease as hereinbefore described in one or more pharmaceutically acceptable carriers and/or diluents.

56. A nucleotide sequence substantially as hereinbefore described with reference to the examples.