WO2004046380A1

WO2004046380A1 - Psoriasis diagnostics and therapeutics

Info

Publication number: WO2004046380A1
Application number: PCT/GB2003/005011
Authority: WO
Inventors: Michael J. Cork; Simon J. Ward; Rachid Tazi-Ahnini
Original assignee: Molecular Skincare Limited
Priority date: 2002-11-20
Filing date: 2003-11-19
Publication date: 2004-06-03
Also published as: AU2003283606A1; GB0227026D0

Abstract

This invention relates to polymorphisms in the human epidermal gene cluster in the major histocompatability complex. In particular, the invention relates to four polymorphisms in the sequence of the α-helix coiled-coil rod homologue (HCR) gene, the structure of the corresponding allelic polypeptides encoded thereby, and the similarity of these polypeptides with streptococcal surface antigen M proteins. The invention provides diagnostic assays for, and methods of manufacture of medicaments for both prophylaxis and treatment of guttate psoriasis and/or psoriasis vulgaris.

Description

PSORIASIS DIAGNOSTICS AND THERAPEUTICS

Field of the Invention

The overall inventive concept of the current invention relates to polymorphisms in the human epidermal gene cluster in the major histocompatability complex. In particular, the invention relates to four polymorphisms in the sequence of the α-helix coiled-coil rod homologue (HCR) gene, the structure of the corresponding allelic polypeptides encoded thereby, and the similarity of these polypeptides with streptococcal surface antigen M proteins. The overall inventive concept gives rise to opportunities for diagnostic assays for, and methods of manufacture of medicaments for both prophylaxis and treatment of guttate psoriasis and/or psoriasis vulgaris.

State of the Art Known to the Applicant

Psoriasis is a common genetically determined inflammatory and proliferative disease of the skin. The most characteristic lesions consist of sharply demarcated, dull-red scaly plaques. The commonest sites that are affected are the knees, elbows, lower back and scalp, although any area of the body may be involved. The commonest clinical form of psoriasis is chronic plaque psoriasis. The plaques of psoriasis vary in diameter from one to several centimetres and are oval or irregular in shape. Chronic plaque psoriasis is the commonest from of psoriasis in adult life.

Guttate psoriasis is a clinical subtype of psoriasis which is characterised by showers of small round lesions of diameter 2mm to 1cm. It occurs most commonly in children and young adults following a streptococcal sore throat. Approximately 70% of individuals who have an episode of guttate psoriasis go on to develop chronic plaque psoriasis.

Guttate plaques of psoriasis erupt 1-2 weeks after an episode of acute streptococcal tonsillitis or pharyngitis. Streptococcal infection may also play a role in the pathogenesis of chronic plaque psoriasis (Wardrop et al., 1998; Tervaert and Esseveld 1970).

The psoriatic lesion is characterised by epidermal hyperproliferation, abnormal differentiation and a mixed mononuclear and neutrophil inflammatory response. There has been much debate as to whether psoriasis is primarily a keratinocyte mediated or T-cell mediated disease. A unifying hypothesis is that an auto antigen is generated within the skin, which leads to an autoimmune inflammatory infiltrate into the skin. The pro-inflammatory cytokines released by T-cells subsequently induce the hyperproliferative changes in the epidermis.

Although there has been much speculation as to the nature of the auto antigen in psoriasis, it has not previously been possible to identify the change or changes in skin associated proteins which lead to the development of an auto antigen.

Infection with group A streptococci may evoke auto immune diseases in susceptible individuals and can affect the skin, heart, kidney, joints and brain. The major virulence factor of these organisms appears to be the surface antigen M proteins, which are a family of closely related proteins emanating from the cell surface as alpha helical coil fibrils. Several studies have demonstrated that purified streptococcal M-proteins stimulate non-immune human T-cells to undergo rapid proliferation. The streptococcal M proteins appear to activate a large proportion of T-cells (circa 20%) not using classical T-cell epitopes. The streptococcal M-proteins are therefore acting as superantigens.

The superantigenic, streptococcus M-proteins induce polyclonal activation of T-cells which then release cytokines such as IL-2 which have been implicated in the development of psoriatic lesions (Skov et al., 1995). Streptococcal superantigens can also induce cutaneous lymphocyte antigen (CLA) on T-cells, thereby facilitating their selective migration from dermal capillaries into the dermis (Baker et al., 1997). It has been shown that uninvolved skin from psoriasis patients grafted onto severe combined immuno-defϊcient mice will develop psoriasis when injected with autologous superantigen treated leucocytes. T-cell lines isolated from psoriatic plaques showed strong reactivity to streptococcal antigens in particular 20 amino acid streptococcal M peptides that share sequences with keratins. It has been postulated that psoriasis may be induced in susceptible individuals by M-protein specific T-cells that cross-react with human type 1 keratin (Valdimarsson et al., 1997).

It has now been established that β-haemolytic streptococci can produce at least two types of exotoxins that can act as superantigens (SAgs). Clinical experience of scarlet fever indicates that streptococcal SAgs can become localized in the skin as a result of streptococcal throat infections, and this is perhaps most clearly demonstrated by the fact that intradermal injection of antisera to the toxins can prevent or abolish the skin rash that is associated with scarlet fever. Using mAbs to nine T-cell receptor (TCR) Vβ2 molecules, a marked increase in the number of Vβ2 T cells, compared with paired blood samples, was demonstrated both in acute and chronic psoriatic skin lesions (Lewis et al.,1993).. A similar, but less marked, over-representation was observed in Vβ5.1 cells, and Vβ8 and Vβl2 T cells were also increased in a few of the patients. Furthermore, an over-representation of Vβ2 was observed in five out if seven T-cells obtained from acute psoriatic lesions, and one line showed an increase in Vβ5.1 and Vβl2. It should be noted that streptococcal superantigens have been reported to react with TCR families that include Vβ2, Vβ5.1, Vβ8 and Vβl2 (Abe et al., 1991).

In scarlet fever the skin rash fades within 2 weeks, whereas guttate psoriasis lesions, which also appear a few days after the throat infection, usually persist for many years. Skin determinant(s) that cross-react with the conventional streptococcal antigen might therefore be a part of the psoriatic predisposition.

In more than 20 different Keratins are encoded in humans by at least as many differentially expressed genes, and this genetic polymorphism may be further increased by allelic variations that have recently been reported, but are as yet largely unexplored. Keratin has a coiled-coil structure similar to streptococcal M-protein (Manjula et al. 1985) and an extensive amino acid sequence homology with the K14 typel human epidermal keratin has been reported (McFadden et al., 1991). Increased expression of K14 and also of K6, K16 and K17 is found in psoriatic patients (Leigh et al., 1995). It is therefore conceivable that the predisposition to psoriasis includes a special allelic variant, or a mutation, of one or more of the keratin genes that are preferentially expressed by hyperproliferating keratinocytes, such 'psoriatic' keratin could have a determinant that is particularly strongly cross-reactive with M-protein, possibly because it is more efficiently presented. Moreover, the expression of this keratin might be induced by cytokines from T cells stimulated by superantigens, conventional antigens or even by the trauma-induced Kobner phenomenon. Thus, the expression of K16 and K17, which is minimal in normal epidermis, is markedly increased during healing. Elevated levels of antibodies to K14 have been reported in patients with psoriasis, indicating that this intracellular self-protein can be recognized by the immune system. These antibodies fluctuate with disease activity, and they are lower in patients with other skin diseases (Aoki et al., 1989). Furthermore, an autoreactivity between epidermal cells and blood lymphocytes has been reported in patients with lichen planus, another putative T- cell medicated skin disease (Steinmuller et al., 1988). In psoriatic lesions, Thl type of cytokine pattern has been demonstrated, and T-cell lines derived from psoriatic lesions were found to produce predominantly Thl-like cytokines (Schlaak et al., 1994), but a similar frequency of Thl- and Th2-like clones was reported, with the majority of clones producing low amount of both IFN- γ and IL-4 (Barna et al., 1991).

However it is not clear if the antibodies and specific T cell clones to keratin 14 may be occurring secondary to the disease process rather than as a primary event which leads to the development of psoriasis. With regard to the genetic hypothesis that an allelic variant of one or more of the keratin genes has been associated with psoriasis, no such genetic associations have been demonstrated.

In 1985 Hensler and Christophers defined two epidermiolgical subtypes of psoriasis. Type 1 age at onset <30 positive family history of psoriasis.

Type 2 age at onset >30 no family history of psoriasis.

They also demonstrated using serological markers that there was a strong association of Type 1 psoriasis with HLACW6, Type 2 psoriasis was not associated with HLACW6.

It had previously been demonstrated that patients with guttate psoriasis had a stronger association with HLACW6 72.7% compared with 45.8% for chronic plaque psoriasis. In the patients with guttate psoriasis those that were CW6 positive had a mean age at onset ofl3.0 ± 4.9 years and those who were CW6 negative had a mean age at onset of 34.8 + 17.8years. A third epidemiological subgroup of psoriasis was defined by Swanbeck et al 1995, this occurred around the age of 14. Enerback et al., (1997) demonstrated that 77% of patients with psoriasis with an age at onset of <22 are cw6 positive. In psoriasis patients who had an age at onset of >22, 38% were cw6 positive. The strongest association of cw6 had a peak age of 14 (Enerback et a.l., 1997)

By 1996 the following had been established;

Guttate psoriasis is commonest in teenagers and young adults.

- A third peak of age of onset of psoriasis occurs around the age of 14. - There was a strong association of Type 1 chronic plaque psoriasis with HLA-C W6

- There was a stronger association of guttate psoriasis with HLA-CW6 and the mean age at onset was 13.

As the strongest candidate marker, the HLA-C gene has been well studied with respect to its involvement in the development of psoriasis (Asahina et al., 1991; Mallon et al., 1997). The strongest HLA-C association has been shown to be with the earliest onset disease type described here as a type la, age at onset (AAO) <20 years, which suggests that early onset psoriasis has a stronger component (Russell et al., 1972; Swanbeck et al., 1995). However, specific involvement of the HLC-Cw6 genotype in the disease pathogenesis has not yet been established, and other candidate genes or loci within the MHC region cannot be excluded. A recent study supports the hypothesis that a major psoriasis predisposing locus resides around the HLA-C region but is probably different from the HLA-C gene itself (Jenisch et al., 1998).

The MHC S gene was initially identified as a 2.6 kb cDNA from a human foetal skin library probed with genomic DNA derived from a YAC clone spanning the HLA-C region. This gene predicts a 488 amino acid protein that maps to a region approximately 160 kb telomeric of the HLA-C locus (Zhou and Chaplin 1993). It is expressed specifically in keratinocytes in the terminal phase of differentiation. Recently, amino acid sequencing of a partially purified 52-56 kDa protein, designated corneodesmosin cDNA (Simon et al., 1997).

Brief Description of the Drawings

The invention will now be described with reference to the accompanying drawings, in which Figure 1 is a schematic diagram showing the position of the HCR gene. The MHC is located on chromosome 6, generally indicated by 1, and the diagram shown the location of the MHC, generally indicated by 2, and within this, the EGC (epidermal gene cluster), generally indicated by 3. Within the MHC-EGC is the HCR gene, generally indicated by 4.

Figure 2 shows the higher structure of the secondary structure of HCR and the Streptococcus pyogenes M24-protein.

Figure 3 shows Western blots performed on total proteins extracted from epidermis using guttate patient serum and compared to HCR polyclonal antibody.

Figure 4 shows Western blots performed on total proteins extracted from epidermis using the serum from members of the same family that suffered from guttate psoriasis. The pattern of binding of the antibodies in these serums was compared to that seen for HCR. Legends to the lanes in the blots are as Figure 3.

Figure 5 shows Immunostaining performed using the avidin-biotin-peroxidase system (ABC kit from Vector Laboratories, USA) and a HCR polyclonal antibody on different epidermal sections. Epidermis from a normal subject was used as a control. Panels A (top left), B (top right) and C (bottom left), were treated with the HCR antibody, and panel D (Bottom right) shows normal epidermis treated with mouse serum therefore acting as a negative control.

Figure 6 shows the amino acid alignment between an HCR mutant and streptococcal protein M24.

Figure 7 shows the amino acid alignment between an HCR mutant and streptococcal protein M2.1.

Summary of the Invention

In its broadest aspect, the invention provides a method for the diagnosis or detection of a polymorphism in HCR in a human, said method comprising the determination of the sequence of the human HCR gene in at least one polymorphic position of HCR and determining the status of the human by reference to the polymorphism in HCR. Preferably, the method determines a sequence wherein the polymorphic position includes at least two of positions 307, 325, 1723 and 2327 in the HCR gene (SEQ ID1).

More preferably, the single nucleotide polymorphism at position 307 is the presence of T; at position 325 is the presence of T; at position 1723 is the presence of T; and at position 2327 is the presence of G.

More preferably also, the polymorphism comprises the presence of the single nucleotide polymorphisms at positions 1723 and 2327.

More preferably also, the polymorphism additionally comprises the presence of the single nucleotide polymorphisms at positions 307 and 325.

The invention also provides a method of detecting an amino acid polymorphism wherein the said polymorphism comprises the presence of cysteine at positions 575 and 776 in the protein encoded by the HCR gene. Preferably, the polymorphism additionally comprises the presence of tryptophan at positions 103 and 109 in the protein encoded by the HCR gene.

The invention also provides the use of any of the preceding methods for the development of a diagnostic assay for susceptibility and/or predisposition to guttate psoriasis.

Also, the invention provides the use of any of the methods above for the development of a diagnostic assay for susceptibility and/or predisposition to chronic plaque psoriasis.

The scope of the invention also includes diagnostic assays produced according to either of the above methods for development of diagnostic assays.

A further aspect of the invention is the use of a vaccine against streptococcus for the manufacture of a medicament for the prophylactic treatment of guttate psoriasis or psoriasis vulgaris.

Preferably, the vaccine is targeted to streptococcal M protein. More preferably, the streptococcal M protein is M21 (SEQ ID6). More preferably also, the streptococcal M protein is M24 (SEQ ID5). Also more preferably, the vaccine is targeted to a combination of streptococcal M protein M21 and M24 Included within the scope of the invention is the use of the polymorphisms described above for the development of a medicament for the treatment of guttate psoriasis or psoriasis vulgaris.

Preferably, the medicament interferes with the antigenic properties of the polymorphic protein.

Background to the Inventive Concept

In view of the strong genetic association between the MHC and psoriasis, the close proximity of the MHC S gene to HLA-C, and the potential functional importance of the MHC S gene in keratinocyte differentiation, the MHC S gene was investigated for genetic association with this disease. Two polymorphisms in the gene were analysed at positions +619 and +1243 in 235 psoriatic patients and 374 matched controls.

We have shown a strong genetic association between genes in the major histocompatibility complex (MHC) S or comeodesmosin (CDSN) gene (Tazi-Ahnini et al, 1999). We recently established the organisation of a region of 46.4 kb harbouring the CDSN gene. This region contains four other genes, α-helix coiled-coil rod homologue (HCR), small rich proline (SPR1), STG and SEEK1. We called this region the Epidermal Gene Cluster (MHC-EGC) (Figure 1).

We recently showed that there is high-risk haplotypes (HRH) containing 49 SNPs within HCR, SPR1, SEEK1, CDSN and STG using Transmission disequilirium test (TDT) in families with psoriasis vulgaris (RTA, unpublished). There is very strong linkage disequilibrium (LD) within the MHC-EGC suggesting that the association we found between the CDSN and guttate psoriasis could be due to the LD of the CDSN and the true gene within the MHC-EGC. We used protein sequence databases to search for similarity between amino acid sequences encoded by the MHC- EGC genes to streptococcus proteins. Unexpectedly, there is significant amino acid similarity between HCR and streptococcal M proteins, suggesting that streptococcal proteins may mimic HCR and/or HCR peptides that may induce autoimmune reaction in guttate psoriasis patients infected by streptococcus observed in guttate psoriasis lesions. Previous studies showed that patients with guttate psoriasis have IgG antibodies that recognise skin proteins.

Figure 6 shows the amino acid alignment between an HCR mutant and a streptococcal M protein M24. The top line of the alignment shows the amino acid sequence of the HCR mutant (SEQ ID4) and the bottom line shows the amino acid sequence of the streptococcal M24 protein (SEQ ID 5). The dashes in the M24 sequence merely indicate that the sequence has been broken up to illustrate the alignment. The amino acid numbering of the M24 sequence is indicated below the bottom line. Each of the marker dots on the sequence indicates amino acid identity between residues on the HCR mutant and the M24 streptococcal protein. It will be readily apparent to those skilled in the field that the remarkable degree of point homology between the sequences is indicative that, following folding of each of the proteins, a large number of common bases can be present at the same or similar positions in the higher structure, leading to common antigenic properties.

Figure 7 shows a similar alignment, but this time between the HCR mutant (SEQ ID 4) and the streptococcal M2.1 protein (SEQ ID 6). Again, there is considerable point homology between the two sequences, indicative of common antigenic properties of the folded proteins.

The inventive concept is that there is a gene within the MHC-EGC that encodes a protein resembling streptococcal surface protein, resulting in cross-reaction between streptococcal and human skin proteins. The evidence for this is from a genetic and proteomic analysis of HCR and M proteins.

1) Genetic study

HCR is very polymorphic and strongly associated with psoriasis vulgaris (Asumalahti et al, 2000; 2002). This study showed polymorphisms in the HCR gene as follows: HCR 307 CAT, 325 C/T, 1723 G/T and 2327 C/G. (i.e. the wild-type C was replaced by T at position 307, and similarly for the three other positions). This gives amino acid change 103R/W, 109 R/W, 575 G/C and 776 S/C respectively. (Amino acid changes are expressed with standard IUPAC one-letter nomenclature, i.e.R=arginine; W=tryptophan; G=glycine; S=serine, and C=cysteine). They defined psoriasis vulgaris conserved HCR haplotype HCR 307T, HCR 325 T, HCR 1723 T and HCR 2327G which encodes HCR 103W, 109W, 575C and 776C (Asumalahti et al, 2000, 2002). We have demonstrated that both 575C and 776C are strongly associated with guttate psoriasis in case-control study. This association is stronger than the one we found with the CDSN in our original work (Tazi-Ahnini et al, 1999). Allelic distribution of HCR G1723T polymorphism

The following tables show the allelic distribution of the G1723T polymorphism for the two homozygous cases of G/G and T/T, and for the heterozygous case of G/T. The occurrence is shown for control subjects; for subjects with guttate psoriasis, and for subjects with chronic plaque psoriasis.

G/G G/T T/T

Controls 101 62 15

Guttate.psoriasis 10 47 16

Chronic plaque psoriasis 22 58 17

Statistical analysis:

2x3 table:

Control vs guttate psoriasis: Chi Square = 39.73 and pO.OOOl

Control vs chronic plaques: Chi Square = 29.72 and pO.OOOl

2x2 table: grouping GT and GG individuals

G/G GT/TT

Controls 101 77

Guttate.psoriasis 10 63

Chronic plaque psoriasis 22 75

Control vs guttate psoriasis: Chi Square - 38.88 and pO.OOOl; OR= 8.26 (3.98, 17.15) Control vs chronic plaques: Chi Square = 30.10 and pO.OOOl; OR= 4.47 (2.55, 7.83) Odds ratio (OR) for the HCR 1723T is higher in guttate patients OR= 8.26 (3.98, 17.15) compared to chronic plaque psoriasis OR= 4.47 (2.55, 7.83) Allelic distribution of HCRC2327G polymorphism

C/C C/G G/G

Controls 16 93 19

Guttate.psoriasis 7 53 11

Chronic plaque psoriasis 20 57 13

Statistical analysis:

2x3 table:

Control vs guttate psoriasis: Chi Square= 22.11 and p .0001

Control vs chronic plaques: Chi Square = 9.01 and p= 0.0111

2x2 table: grouping GT and GG individuals

C/C CG/GG

Controls 76 112

Guttate.psoriasis 7 64

Chronic plaque psoriasis 20 70

Control vs guttate psoriasis: Chi Square= 38.88 and pO.OOOl; OR= 6.20 (2.70, 14.27) Control vs chronic plaques: Chi Square= 8.92 and p=0.0028; OR= 2.38 (1.34, 4.23)

Chronic plaque vs guttate psorisis: Chi Square= 4.35 and p= 0.0371, OR= 2.6 (1.04, 6.59)

Odds ratio (OR) for the HCR 1227G is higher in guttate patients OR= 6.20 (2.70, 14.27) compared to chronic plaque psoriasis OR= 2.38 (1.34, 4.23)

Moreover, there is significant difference between guttate and chronic plaque psoriasis. Within psoriasis group G at position 2327 of the HCR confers 2.6 more risk to develop guttate psoriasis than chronic plaque psoriasis. It will be apparent to those skilled in the art that this provides the basis for differentiating between guttate psoriasis and chronic plaque psoriasis. 2) In sttico cloning - Modelling of HCR and the Streptococcus M-Protein

The secondary structure of HCR and the Streptococcus pyogenes M24-protein was modelled using the Swiss-Model software on the ExPASy (Expert Protein Analysis System) proteomics server of the Swiss Institute of Bioinformatics (SIB).

Proteins which had the greatest sequence identity to the amino acid sequences of HCR and the M24-protein were identified using the BLAST software from NCBI. These proteins were used as a template for modelling HCR and the M-protein. The structures derived by the software are shown in Figure 2.

For modelling HCR the Fibritin Deletion Mutant M protein was used as a template and for the M- protein the Rat Tropomyosin was used. Both models show that HCR and the M24 protein have a helix coiled structure.

3) Proteomic study

Amino acid sequence analysis of HCR (SEQ ID 3) reveals that this protein belongs to the nuclear transcription factor family. The four mutations C307T, C325T, C1723T and C2327G in the HCR gene give an amino acid change from R103W, R109W, G575C and S776C respectively (SEQ ID 4). Amino acids at positions 103, 109, 575 and 776 are within the region containing LXXLL motifs that are known to interact with nuclear receptors. Substitution of R103, R109, G575 and S776 to W103, W109, C575 and C776 respectively may change the affinity of HCR protein to interact with nuclear receptors. To verify this hypothesis we have expressed in-vitro the HCR containing either R103, R109, G575 and S776 (wild-type) or W103, W109, C575 and C776 (mutant) amino acids. Using indirect immunofluorescence analysis in COS-1 cells transiently transfected with either HCR R103, R109, G575 and S776 (wild-type) or W103, W109, C575 and C776 (mutant) expression constructs we will be able the subcellular locations of mutant and wild type forms of HCR protein.

Antibody Production

Monoclonal and polyclonal antibodies were generated by the Antibody Resource Centre, at the University of Sheffield. The peptides used for antibody production are marked on the HCR sequence (SEQ ID 3) below. 4 rabbits and 4 mice were immunised with peptide 571- Thyroglobulin (THY) conjugate, 4 rabbits and 4 mice with 572-THY and 4 rabbits and 4 mice with 588-THY. For 571 monoclonal antibody, fusion was made using spleen from immunised mice and plated into 10 x 96 well plates. A total of 22 colonies were screened by ELISA and western blot. Positive clones were ampoules frozen. Similar approach was used to screen 572 and 588 antibodies.

Antibodies were generated such that they would recognise and bind HCR (HCR588, a polyclonal antibody) and (HCR571 and HCR572 are polyclonal and monoclonal antibodies) of the protein.

Candidate antibodies were screened by a standard western blotting method using total protein extracts from control subject epidermis.

HCR Wild Type Amino Acid Sequence (SEQ ID 3)

Peptides HCR572, HCR571 and HCR588 are shown respectively :-

HCR572

MFPPSGSTGL IPPSHFQARP LSTLPRMAPT WLSDIPLV OPPGHODVSERR DTQRPQVTM WERDVSSDRQ EPGRRGRSWG LEGSQALSQQ AEVIVRQLQE LRRLEEEVRL LRETSLQQKM RLEAQAMELE ALARAEKAGR AEAEGLRAAL

HCR571 AGAEVV RKNLEEGRO RELEE VQRLHQEQLS SLTQAHEEAL SSLTSKAEGL EKSLSSLETR RAGEAKELAE AQREAELLRK QLSKTQEDLE AQVTLVENLR KYVGEQVPSE VHSQTWELER QKLLDTMQHL QEDRDSLHAT AELLQVRVQS LTHILALQEE ELTRKVQPSD SLEPEFTRKC QSLLNRWREK VFALMVQLKA QELEHSDSVK QLKGQVASLQ EKVTSQSQEQ AILQRSLQDK AAEVEVERMG AKGLQLELSR AQEARRRWQQ QTASAEEQLR LVVNAVSSSQ IWLETTMAKV EGAAAQLPSL NNRLSYAVRK VHTIRGLIAR KLALAQLRQE SCPLPPPVTD VSLELQQLRE ERNRLDAELQ LSARLIQQEV GRAREQGEAE RQQLSKVAQQ LEQELQQTQE SLASLGLQLE VARQGQQEST EEAASLRQEL TQQQELYGQA LQEKVAEVET RLREQLSDTE RRLNEARREH AKAVVSLRQI QRRAAQEKER

HCR588 SOELRRLOEEAR KEEGQRLA RRLQELERDK NLMLATLQQE GLLSRYKQQR LLTVLPSLLD KKKSVVSSPR PPECSASAPV AAAVPTRESI KGSLSVLLDD LQDLSEAISK EEAVCQGDNL DRCSSSNPQM SS^» Western Blots With Index Guttate Patient Serum

Nitrocellulose membranes produced with total protein (20μg of total protein) from:- Normal control epidermis Psoriaitc Lesional epidermis

Psoriaitc Non-Lesional epidermis Proliferating Keratinocytes (PKC) Differentiating Keratinocytes (DKC) Standard western blot procedure followed. Membranes incubated with serum from guttate patients or with HCR polyclonal antibody at a 1:1000 dilution.

Western blots were performed using guttate patient serum and compared to HCR polyclonal antibody. Figure 3 shows that the antibodies in the guttate serum and the HCR polyclonal antibody bound similar proteins in the 5 different extracts listed above. There are HCR antibodies in serum of patients with guttate psoriasis. We also used serum of controls with no history of psoriasis on western blot and found negative reaction (data not shown). These results suggest that there is autoimmune reaction develop against HCR protein in guttate psoriasis patients who had streptococcal infection.

Eleven different guttate patient serums were studied by western blotting. Of these, 8 bound to epidermal proteins.

Western Blots With serums of Guttate Patient and his parent

Western blots were performed using the serum from members of the same family that suffered from guttate psoriasis. The pattern of binding of the antibodies in these serums was compared to that seen for HCR.

As can be seen in figure 4, one of the patient serums (B) bound to proteins around the 70-85 kDa mark in all 3 protein extracts, normal epidermis, psoriatic lesionsal and non-lesionsal. This pattern was similar to that seen in the HCR western blot (figure 3). The other serum (serum A) showed no binding at all (patient's parent). This indicates that there is a difference in the serums of parent and off-spring could be due to disease manifestation. Western Blots With Control Subject Serum

A negative control was performed to establish if the binding seen between guttate patient serum and proteins from the dermal samples was specific and exclusive to this patient sub-group or non- specific.

Western blots were performed using the serum from control subjects. There was no binding between the control serum and the epidermal proteins (data not shown).

HCR immunohistochemistry

Human epidermis was obtained from patients and healthy volunteers from the Royal Hallamshire Hospital, Sheffield, with full ethical consent. Tissue was formalin-fixed and embedded in paraffin. Immunostaining was performed using the avidin-biotin-peroxidase system (ABC kit from Vector Laboratories, USA) and a HCR polyclonal antibody on different epidermal sections. The HCR antibody was used at a 1 : 100 dilution. Epidermis from a normal subject was used as a control. Panels A, B and C, figure 5, were treated with the HCR antibody, and panel D shows normal epidermis treated with mouse serum therefore acting as a negative control.

There is very strong expression of HCR protein psoriatic lesional skin mainly on the basal layer and the stratum corneum. Very weak expression of HCR has been revealed in the stratum corneum and basal layer of normal epidermis. Negative control (Rabbit serum) doesn't show any reactivity. Interestingly, a very strong expression of HCR has been detected on psoriatic non- lesional skin. These results suggest that first high expression of HCR in psoriatic lesional skin is not induced by the inflammation state of the skin. Secondly, HCR is a predisposing factor in psoriatic patient. HCR over-expression is the initiating factor to the formation of the plaque.

Expression of the HCR wild type and mutant Proteins

HCR expression was performed by standard cloning and expression techniques. The HCR construct was cloned into a gWizTag vector and the clone was transfected into COS-7 cells (African green monkey kidney cells) using GeneJuice (Novagen, Germany). HCR expression was confirmed by western blotting using an anti-his tag antibody. Four mutations in HCR coding sequence T at position 307, T at position 325, T at position 1723 and G at position 2327 G. Mutant sequence has been cloned and expressed in COS-7 as described above for Wild type sequence.

Identification of a specific skin protein variant strongly associated with guttate psoriasis would be a major step towards developing novel and specific therapies such as vaccines to prevent attacks of this condition in susceptible individuals.

Furthermore, the association with the haplotypes described above with individuals susceptible to development of guttate psoriasis provides a basis for a diagnostic assay for identifying susceptible individuals. The assay could be embodied in a number of ways, as follows:

The predisposition of susceptibility of individuals to guttate psoriasis may be determined by detecting the presence of the polymorphisms described above by the use of methods known in the art such as polymerase chain reaction; the loss or gain of sites recognised restriction enzymes; Amplification Refractory Mutation System (ARMS) - allele specific amplification; oligonucleotide ligation assay or restriction fragment length polymorphism (RFLP).

Alternatively, the variant protein containing the amino acid polymorphism encoded by the haplotype could be readily detected by immunological methods such as enzyme-linked immunosorbent assay (ELISA).

Furthermore, the invention leads to a novel process for manufacturing a medicament for the prophylaxis of guttate psoriasis and/or chronic plaque psoriasis whereby a vaccine is produced against streptococcus, optionally specifically targeting the M proteins described, and optionally more specifically the M21 (SEQ ID 6) and M24 (SEQ ID 5) proteins. Alternatively, the vaccine may be targeted against the altered HCR protein described above.

Additionally, the invention leads to a novel process for manufacturing a medicament for the treatment of guttate psoriasis and/or chronic plaque psoriasis whereby a therapeutic agent is provided to block the antigenic properties of the variant HCR protein described above.

Furthermore, the overall inventive concept is applicable to diagnostics and therapeutics for other diseases involving the action of HCR-mediated auto immune reactions. The most important disease group is vasculitis. There are many different forms of vasculitis and it may affect virtually every organ in the body. Vasculitis is an inflammatory response around blood vessels. The disease manifestations may vary enormously depending on the degree of inflammation and size of the blood vessels involved. Patients may develop renal failure, ulcers and arthritis. Specific examples include leukocytoclastic vasculitis and Kawasaki's disease.

Sequences

The nucleotide and amino acid sequences for the "normal" and "mutant" HCR gene and expressed polypetides are presented below. The polymorphisms discussed in the text are indicated by bases or amino acids in bold type. Standard nomenclature is used (ATCG for the nucleotide bases, and RJPAC one-letter coding for the polypeptide sequences).

HCR cDNA sequences Wild type HCR: (SEQ ID 1)

ATGTTTCCACCTTCAGGTTCCACTGGGCTGATTCCCCCCTCCCACTTTCAAGCT CGGCCCCTTTCAACTCTGCCAAGAATGGCTCCCACCTGGCTCTCAGACATTCC CCTGGTCCAACCCCCAGGCCATCAAGATGTCTCAGAGAGGCGGCTAGACACC CAGAGACCTCAAGTGACCATGTGGGAACGGGATGTTTCCAGTGACAGGCAGG AGCCAGGGCGGAGAGGCAGGTCCTGGGGGCTGGAGGGGTCACAGGCCCTGA

GCCAGCAGGCTGAGGTGATCGTTCGGCAGCTGCAAGAGCTGCGGCGGCTGG

AGGAGGAGGTCCGGCTCCTGCGGGAGACCTCGCTGCAGCAGAAGATGAGGC TAGAGGCCCAGGCCATGGAGCTAGAGGCTCTGGCACGGGCGGAGAAGGCCG GCCGAGCTGAGGCTGAGGGCCTGCGTGCTGCTTTGGCTGGGGCTGAGGTTGT CCGGAAGAACTTGGAAGAGGGGAGGCAGCGGGAGCTGGAAGAGGTTCAGAG GCTGCACCAAGAGCAGCTGTCCTCTTTGACACAGGCTCACGAGGAGGCTCTT TCCAGTTTGACCAGCAAGGCTGAGGGCTTGGAGAAGTCTCTGAGTAGTCTGG AAACCAGAAGAGCAGGGGAAGCCAAGGAGCTGGCCGAGGCTCAGAGGGAG GCCGAGCTGCTTCGGAAGCAGCTGAGCAAGACCCAGGAAGACTTGGAGGCTC AGGTGACCCTGGTTGAGAATCTAAGAAAATATGTTGGGGAACAAGTCCCTTC TGAGGTCCACAGCCAGACATGGGAACTGGAGCGACAGAAGCTTCTGGACACC ATGCAGCACTTGCAGGAGGACCGGGACAGCCTGCATGCCACCGCGGAGCTGC TGCAGGTGCGGGTGCAGAGCCTCACACACATCCTCGCCCTGCAGGAGGAGGA GCTGACCAGGAAGGTTCAACCTTCAGATTCCCTGGAGCCTGAGTTTACCAGG AAGTGCCAGTCCCTGCTGAACCGCTGGCGGGAGAAGGTGTTTGCCCTCATGG TGCAGCTAAAGGCCCAGGAGCTGGAACACAGTGACTCTGTTAAGCAGCTGAA GGGACAGGTGGCCTCACTCCAGGAAAAAGTGACATCCCAGAGCCAGGAGCA GGCCATCCTGCAGCGATCCCTGCAGGACAAAGCCGCAGAGGTGGAGGTGGA GCGTATGGGTGCCAAGGGCCTGCAGTTGGAGCTGAGCCGTGCTCAGGAGGCC AGGCGTCGGTGGCAGCAGCAGACAGCCTCAGCCGAGGAGCAGCTGAGGCTT GTGGTCAATGCTGTCAGCAGCTCTCAGATCTGGCTCGAGACCACCATGGCTA AGGTGGAAGGGGCTGCCGCCCAGCTTCCCAGCCTCAACAACCGACTCAGCTA TGCTGTCCGCAAGGTCCACACCATTCGGGGCCTGATTGCTCGAAAGCTTGCCC TTGCTCAGCTGCGCCAGGAGAGCTGTCCCCTACCACCACCGGTCACAGATGT GAGCCTTGAGTTGCAGCAGCTGCGGGAAGAACGGAACCGCCTGGATGCAGA ACTGCAGCTGAGTGCCCGCCTCATCCAGCAGGAGGTGGGCCGGGCTCGGGAG CAAGGGGAGGCAGAGCGGCAGCAGCTGAGCAAGGTGGCCCAGCAGCTGGAG CAGGAGCTGCAGCAGACCCAGGAGTCCCTGGCTAGCTTGGGGCTGCAGCTGG

AGGTAGCACGCCAGGGCCAGCAGGAGAGCACAGAGGAGGCTGCCAGTCTGC GGCAGGAGCTGACCCAGCAGCAGGAACTCTACGGGCAAGCCCTGCAAGAAA AGGTGGCTGAAGTGGAAACTCGGCTGCGGGAGCAACTCTCAGACACAGAGA GGAGGCTGAACGAGGCTCGGAGGGAGCATGCCAAG:GCCGTGGTCTCCTTGC GCCAGATTCAGCGCAGAGCCGCCCAGGAAAAGGAGCGGAGCCAGGAACTCA GGCGTCTGCAGGAGGAGGCCCGGAAGGAGGAGGGGCAGCGACTGGCCCGGC GCTTGCAGGAGCTAGAGAGGGATAAGAACCTCATGCTGGCCACCTTGCAGCA GGAAGGTCTCCTCTCCCGTTACAAGCAGCAGCGACTGTTGACAGTTCTTCCTT CCCTACTGGATAAGAAGAAATCTGTGGTGTCCAGCCCCAGGCCTCCAGAGTG TTCAGCATCTGCACCTGTAGCAGCAGCAGTGCCCACCAGGGAGTCCATAAAA GGGTCCCTCTCTGTCCTGCTCGATGACCTGCAGGACCTGAGTGAAGCCATTTC

CAAAGAGGAAGCTGTTTGTCAAGGAGACAACCTTGACAGATGCTCCAGCTCC AATCCCCAGATGAG

Mutant HCR (SEQ ID 2)

GCCAGCAGGCTGAGGTGATCGTTCGGCAGCTGCAAGAGCTGCGGTGGCTGG AGGAGGAGGTCTGGCTCCTGCGGGAGACCTCGCTGCAGCAGAAGATGAGGC TAGAGGCCCAGGCCATGGAGCTAGAGGCTCTGGCACGGGCGGAGAAGGCCG GCCGAGCTGAGGCTGAGGGCCTGCGTGCTGCTTTGGCTGGGGCTGAGGTTGT CCGGAAGAACTTGGAAGAGGGGAGGCAGCGGGAGCTGGAAGAGGTTCAGAG GCTGCACCAAGAGCAGCTGTCCTCTTTGACACAGGCTCACGAGGAGGCTCTT TCCAGTTTGACCAGCAAGGCTGAGGGCTTGGAGAAGTCTCTGAGTAGTCTGG AAACCAGAAGAGCAGGGGAAGCCAAGGAGCTGGCCGAGGCTCAGAGGGAG GCCGAGCTGCTTCGGAAGCAGCTGAGCAAGACCCAGGAAGACTTGGAGGCTC AGGTGACCCTGGTTGAGAATCTAAGAAAATATGTTGGGGAACAAGTCCCTTC TGAGGTCCACAGCCAGACATGGGAACTGGAGCGACAGAAGCTTCTGGACACC ATGCAGCACTTGCAGGAGGACCGGGACAGCCTGCATGCCACCGCGGAGCTGC TGCAGGTGCGGGTGCAGAGCCTCACACACATCCTCGCCCTGCAGGAGGAGGA GCTGACCAGGAAGGTTCAACCTTCAGATTCCCTGGAGCCTGAGTTTACCAGG AAGTGCCAGTCCCTGCTGAACCGCTGGCGGGAGAAGGTGTTTGCCCTCATGG TGCAGCTAAAGGCCCAGGAGCTGGAACACAGTGACTCTGTTAAGCAGCTGAA GGGACAGGTGGCCTCACTCCAGGAAAAAGTGACATCCCAGAGCCAGGAGCA GGCCATCCTGCAGCGATCCCTGCAGGACAAAGCCGCAGAGGTGGAGGTGGA GCGTATGGGTGCCAAGGGCCTGCAGTTGGAGCTGAGCCGTGCTCAGGAGGCC AGGCGTCGGTGGCAGCAGCAGACAGCCTCAGCCGAGGAGCAGCTGAGGCTT GTGGTCAATGCTGTCAGCAGCTCTCAGATCTGGCTCGAGACCACCATGGCTA AGGTGGAAGGGGCTGCCGCCCAGCTTCCCAGCCTCAACAACCGACTCAGCTA TGCTGTCCGCAAGGTCCACACCATTCGGGGCCTGATTGCTCGAAAGCTTGCCC TTGCTCAGCTGCGCCAGGAGAGCTGTCCCCTACCACCACCGGTCACAGATGT GAGCCTTGAGTTGCAGCAGCTGCGGGAAGAACGGAACCGCCTGGATGCAGA ACTGCAGCTGAGTGCCCGCCTCATCCAGCAGGAGGTGGGCCGGGCTCGGGAG CAAGGGGAGGCAGAGCGGCAGCAGCTGAGCAAGGTGGCCCAGCAGCTGGAG CAGGAGCTGCAGCAGACCCAGGAGTCCCTGGCTAGCTTGGGGCTGCAGCTGG

AGGTAGCACGCCAGTGCCAGCAGGAGAGCACAGAGGAGGCTGCCAGTCTGC GGCAGGAGCTGACCCAGCAGCAGGAACTCTACGGGCAAGCCCTGCAAGAAA AGGTGGCTGAAGTGGAAACTCGGCTGCGGGAGCAACTCTCAGACACAGAGA GGAGGCTGAACGAGGCTCGGAGGGAGCATGCCAAG:GCCGTGGTCTCCTTGC GCCAGATTCAGCGCAGAGCCGCCCAGGAAAAGGAGCGGAGCCAGGAACTCA GGCGTCTGCAGGAGGAGGCCCGGAAGGAGGAGGGGCAGCGACTGGCCCGGC GCTTGCAGGAGCTAGAGAGGGATAAGAACCTCATGCTGGCCACCTTGCAGCA GGAAGGTCTCCTCTCCCGTTACAAGCAGCAGCGACTGTTGACAGTTCTTCCTT CCCTACTGGATAAGAAGAAATCTGTGGTGTCCAGCCCCAGGCCTCCAGAGTG TTCAGCATCTGCACCTGTAGCAGCAGCAGTGCCCACCAGGGAGTCCATAAAA GGGTCCCTCTCTGTCCTGCTCGATGACCTGCAGGACCTGAGTGAAGCCATTTC

CAAAGAGGAAGCTGTTTGTCAAGGAGACAACCTTGACAGATGCTCCAGCTG CAATCCCCAGATGAG

HCR amino acid sequences

Wild type HCR (SEQ ID 3)

MFPPSGSTGLIPPSHFQARPLSTLPRMAPTWLSDIPLVQPPGHQDVSERRLDTQRPQ VTMWERDVSSDRQEPGRRGRSWGLEGSQALSQQAEVIVRQLQELRR₁₀₃LEEEV R₁₀9LLRETSLQQKMRLEAQAMELEALARAEKAGRAEAEGLRAALAGAEVVRKNL EEGRQRELEEVQRLHQEQLSSLTQAHEEALSSLTSKAEGLEKSLSSLETRRAGEAK ELAEAQREAELLRKQLSKTQEDLEAQVTLVENLRKYVGEQVPSEVHSQTWELERQ KLLDTMQHLQEDRDSLHATAELLQVRVQSLTHILALQEEELTRKVQPSDSLEPEFT RKCQSLLNRWREKVFALMVQLKAQELEHSDSVKQLKGQVASLQEKVTSQSQEQA ILQRSLQDKAAEVEVERMGAKGLQLELSRAQEARRRWQQQTASAEEQLRLVVNA VSSSQIWLETTMAKVEGAAAQLPSLNNRLSYAVRKVHTIRGLIARKLALAQLRQE SCPLPPPVTDVSLELQQLREERNRLDAELQLSARLIQQEVGRAREQGEAERQQLSK VAQQLEQELQQTQESLASLGLQLEVARQG₅₇₅QQESTEEAASLRQELTQQQELYGQ ALQEKVAEVETRLREQLSDTERRLNEARREHAKAVVSLRQIQRRAAQEKERSQEL RRLQEEARKEEGQRLARRLQELERDKNLMLATLQQEGLLSRYKQQRLLTVLPSLL DKKKSVVSSPRPPECSASAPVAAAVPTRESIKGSLSVLLDDLQDLSEAISKEEAVCQ GDNLDRCSSS₇₇₆NPQMSS^«

Mutant HCR (SEQ ID 4)

MFPPSGSTGLIPPSHFQARPLSTLPRMAPTWLSDIPLVQPPGHQDVSERRLDTQRPQ VTMWERDVSSDRQEPGRRGRSWGLEGSQALSQQAEVIVRQLQELRW₁₀₃LEEEV W₁₀9LLRETSLQQKMRLEAQAMELEALARAEKAGRAEAEGLRAALAGAEVVRKN LEEGRQRELEEVQRLHQEQLSSLTQAHEEALSSLTSKAEGLEKSLSSLETRRAGEA KELAEAQREAELLRKQLSKTQEDLEAQVTLVENLRKYVGEQVPSEVHSQTWELER QKLLDTMQHLQEDRDSLHATAELLQVRVQSLTHILALQEEELTRKVQPSDSLEPEF TRKCQSLLNRWREKVFALMVQLKAQELEHSDSVKQLKGQVASLQEKVTSQSQEQ AILQRSLQDKAAEVEVERMGAKGLQLELSRAQEARRRWQQQTASAEEQLRLVVN AVSSSQIWLETTMAKVEGAAAQLPSLNNRLSYAVRKVHTIRGLIARKLALAQLRQ ESCPLPPPVTDVSLELQQLREERNRLDAELQLSARLIQQEVGRAREQGEAERQQLS KVAQQLEQELQQTQESLASLGLQLEVARQC₅₇₅QQESTEEAASLRQELTQQQELYG QALQEKVAEVETRLREQLSDTERRLNEARREHAKAVVSLRQIQRRAAQEKERSQE LRRLQEEARKEEGQRLARRLQELERDKNLMLATLQQEGLLSRYKQQRLLTVLPSL LDKKKSVVSSPRPPECSASAPVAAAVPTRESIKGSLSVLLDDLQDLSEAISKEEAVC QGDNLDRCSSC₇₇₆NPQMSS^«

Streptococcal M Protein M24 (Gen Bank Accession Number: P12379) - SEQ ID 5

MTKNNTNRHY SLRKLKTGTA S VAVALTVLG AGLVVNTNEV SAVATRSQTD TLEKVQERAD KFEIENNTLK LKNSDLSFNN KALKDHNDEL TEELSNAKEK LRKNDKSLSE KASKIQELEA RKADLEKALE GAMNFSTADS AKIKTLEAEK AALAARKADL EKALEGAMNF STADSAKIKT LEAEKAALEA RQAELEKALE GAMNFSTADS AKIKTLEAEK AALAARKADL EKALEGAMNF STADSAKIKT LEAEKAALEA RQAELEKALE GAMNFSTADS AKIKTLEAEK AALEAEKADL EHQSQVLNAN RQSLRRDLDA SREAKKQLEA EHQKLEEQNK ISEASRQSLR RDLDASREAK KQLEAEHQKL EEQNKISEAS RQSLRRDLDA SREAKKQVEK ALEEANSKLA ALEKLNKELE ESKKLTEKEK AELQAKLEAE AKALKEKLAK QAEELAKLRA GKASDSQTPD AKPGNKAVPG KGQAPQAGTK PNQNKAPMKE TKRQLPSTGE TANPFFTAAA LTVMATAGVA AVVKRKEEN

Streptococcal M Protein M2.1 (Gen Bank Accession Number: PP50468) - SEQ ID 6 MARKDTNKQY SLRKLKTGTA SVAVAVAVLG AGFANQTTVK ANSKNPVPVK KEAKLSEAEL HDKIKNLEEE KAELFEKLDK VEEEHKKVEE EHKKDHEKLE KKSEDVERHY LRQLDQEYKE QQERQKNLEE LERQSQREVE KRYQEQLQKQ QQLEKEKQIS EASRKSLRRD LEASRAAKKD LEAEHQKLKE EKQISEASRK SLRRDLEASR AAKKDLEAEH QKLKEEKQIS EASRQGLSRD LEASRAAKKD LEAEHQKLKE EKQISEASRQ GLSRDLEASR EAKKKVEADL AEANSKLQAL EKLNKELEEG KKLSEKEKAE LQAKLEAEAK ALKEQLAKQA EELAKLKGNQ TPNAKVAPQA NRSRSAMTQQ KRTLPSTGET ANPFFTAAAA TVMVSAGMLA LKRKEEN

References

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Claims

1. A method for the diagnosis or detection of a polymorphism in HCR in a human, said method comprising the determination of the sequence of the human HCR gene in at least one polymorphic position of HCR and determining the status of the human by reference to the polymorphism in HCR.

2. A method according to claim 1, wherein the polymorphic position includes at least two of positions 307, 325, 1723 and 2327 in the HCR gene.

3. A method according to claim 2 wherein the single nucleotide polymorphism at position 307 is the presence of T; at position 325 is the presence of T; at position 1723 is the presence of T; and at position 2327 is the presence of G.

4. A method according to claim 3 wherein the polymorphism comprises the presence of the single nucleotide polymorphisms at positions 1723 and 2327.

5. A method according to claim 4, wherein the polymorphism additionally comprises the presence of the single nucleotide polymorphisms at positions 307 and 325.

6. A method of detecting an amino acid polymorphism wherein the said polymorphism comprises the presence of cysteine at positions 575 and 776 in the protein encoded by the HCR gene.

7. A method according to claim 6, wherein the polymorphism additionally comprises the presence of tryptophan at positions 103 and 109 in the protein encoded by the HCR gene.

8. The use of any of the preceding methods for the development of a diagnostic assay for susceptibility and/or predisposition to guttate psoriasis.

9. The use of any of the methods 1 to 7 for the development of a diagnostic assay for susceptibility and/or predisposition to chronic plaque psoriasis.

10 A diagnostic assay according to either of claims 8 or 9.

11 The use of a vaccine against streptococcus for the manufacture of a medicament for the prophylactic treatment of guttate psoriasis or psoriasis vulgaris.

12. The use of claim 11 wherein the vaccine is targeted to streptococcal M protein.

13. The use of claim 12 wherein the streptococcal M protein is M21 (SEQ ID 6).

14. The use of claim 12 wherein the streptococcal M protein is M24 (SEQ ID 5).

15. The use of claim 12 wherein the vaccine is targeted to a combination of streptococcal M protein M21 and M24.

16. The use of the polymorphism in any of claims 1 to 6 for the development of a medicament for the treatment of guttate psoriasis or psoriasis vulgaris.

17. The use of claim 16 wherein the medicament interferes with the antigenic properties of the polymorphic protein.