WO2009037295A1

WO2009037295A1 - Method for testing psoriasis susceptibility

Info

Publication number: WO2009037295A1
Application number: PCT/EP2008/062401
Authority: WO
Inventors: Judith Fischer
Original assignee: Commissariat A L'energie Atomique
Priority date: 2007-09-18
Filing date: 2008-09-18
Publication date: 2009-03-26

Abstract

The present invention concerns a method of testing a subject thought to have or be predisposed to having psoriasis which comprises the step of analyzing a biological sample from said subject for i) detecting the presence of a SNP in the ADAM33 gene, which SNP is associated with psoriasis, and/or ii) analyzing the expression of the ADAM33 gene.

Description

METHOD FOR TESTING PSORIASIS SUSCEPTIBILITY

This patent application claims the priority of provisional patent application US 60/973,235 filed on September 18, 2007 which is herein incorporated by reference.

Field of the invention

The invention relates to the field of psoriasis susceptibility, and preferably to a method of testing a subject thought to have or be predisposed to having psoriasis.

Background of the Invention

Psoriasis [MIM 177900] is a common hyperproliferative and chronic inflammatory skin disease with a prevalence of about 2-4% in Caucasian populations [NEVITT & HUTCHINSON, Br. J. Dermatol, vol.135, p:533-537, 1996]. Plaque psoriasis, also known as psoriasis vulgaris, is by far the most common type of psoriasis, accounting for 80%-90% of all psoriasis patients. It appears as raised red scaling patches, most frequently on the elbows, knees, scalp and lower back.

This autoimmune disease is regarded as a multifactorial trait involving environmental factors such as intake of certain drugs, psychosocial stress, smoking, or climate conditions, all of which are well known triggering factors for primary manifestations or exacerbation in susceptible individuals [TAGAMI, Clin. Dermatol., vol.15, p:677-685, 1997]. On the other hand, evidence for a strong genetic component is provided by twin and family studies, which have shown a concordance rate of psoriasis in monozygotic twins of 65-72% vs 15-30% in dizygotic twins and a heritability of 80%

[BOWCOCK & COOKSON, Hum. MoI. Genet., vol.13, p:R43-55, 2004].

In an attempt to elucidate the genetic basis of psoriasis, a number of genome-wide linkage studies have been undertaken. Overwhelming evidence for a susceptibility locus has been found for Chromosome 6p21 within the HLA region [NAIR et al., Hum. MoI. Genet., vol.6, p:349-1356, 1997; TREMBATH et al, Hum. MoI Genet., vol.6, p:813- 820, 1997; SAMUELSSON et al, Hum. Genet., vol.105, p:523-529, 1999 ; ENLUND et al, Hum. Hered., vol.49, p:2-8, 1999; VEAL et al, J. Med. Genet., vol.38, p:7-13, 2001; ZHANG et al, J. Invest. Dermatol, vol.119, p:1361-1366, 2002 ; SAGOO et al, J. Invest. Dermatol, vol.122, p:1401-1405, 2004 ; LESUEUR et al, Journal of Investigative Dermatology, Mar 8 [Epub ahead of print], 2007]. In particular, association studies employing linkage disequilibrium (LD) mapping have been successful in narrowing the locus to a 300kb interval [HEWETT et al, Genomics, vol.79, p: 305-314, 2002; VEAL et al, Am. J. Hum. Genet., vol.71, p:554-564, 2002; LENCH et al, J. Invest. Dermatol, vol.124, p:545-552, 2005 ; HELMS et al, Hum. Genet., vol.118, p:466-476, 2005 ; NAIR et al, Am. J. Hum. Genet., vol.78, p:827-851, 2006] and then in identifying HLA-Cw6 as the disease allele at the 6p21 locus [NAIR et al, abovementionned, 2006 ; ELDER, J. Invest. Dermatol, vol.126, p: 1205-1206, 2006]. This locus, referred as PSORSl (Psoriasis Susceptibility 1, [MIM 177900] contributes to the familial clustering of disease (λ) to 33 < λ < 50% [TREMBATH et al, abovementionned, 1997; The International Psoriasis Genetics Study, Am. J. Hum. Genet., vol.73, p:430-437, 2003]. Therefore, other susceptibility genes are likely to exist. Genome-wide linkage analyses have highlighted a number of disease loci on at least 15 chromosomes (see [LESUEUR et al, abovementionned, 2007] for review). Elucidation of the disease genes in these candidate loci is hampered by their large size and by the large number of candidates in each region.

Summary of the invention

In a first aspect, the present invention relates to a method of testing a subject thought to have or be predisposed to having psoriasis which comprises the step of analyzing a biological sample from said subject for:

i) detecting the presence of a SNP in the ADAM33 gene, which SNP is associated with psoriasis , and/or ii) analyzing the expression of the ADAM33 gene.

In another embodiment, the present invention provides also a method for treating and/or preventing psoriasis in a subject, comprising the administration of an effective amount of a compound which specifically inhibits the expression of ADAM33 gene to said subject.

In still another embodiment, the present invention provides also a method for treating and/or preventing psoriasis in a subject, comprising the administration of an effective amount of a compound which specifically increases the expression of ADAM33 gene to said subject.

In another embodiment, the present invention provides also an in vitro method of selecting a compound, which can be useful for treating psoriasis, characterized in that said method comprises the steps of:

a) obtaining a cell expressing ADAM33 gene; b) contacting said cell with at least one compound, c) comparing the expression of the ADAM33 gene in the cell between the steps a) and b), d) selecting the compound, which induces a lower level of expression of the ADAM33 gene in the cell contacted to that compound.

In still another embodiment, the present invention provides also an in vitro method of selecting a compound, which can be useful for treating psoriasis, characterized in that said method comprises the steps of:

a) obtaining a cell expressing AD AM33 gene;

b) contacting said cell with at least one compound,

c) comparing the expression of the ADAM33 gene in the cell between the steps a) and b),

d) selecting the compound, which induces a higher level of expression of the ADAM33 gene in the cell contacted to that compound.

Brief description of the drawings

Figurel shows the schematic representation of the psoriasis susceptibility locus on Chromosome 20pl3.

Table 1 shows SNPs genotyped at the ADAM33 locus and FBAT results for the univariate analysis. Table 2. Most significant under- and over-transmitted 3-SNP haplotypes for Region 2.

Table 3 shows the comparison of Set I and Set II

Table 4 shows the univariate SNP analysis in Set II and in combined sets. Only SNPs showing some association with psoriasis in Set I, in the univariate or in the 3-SNP haplotype analyses, were tested in Set II.

Supplementary Table Sl shows the results of the association tests for SNPs selected in Stage I.

Supplementary Table S2 shows the results for DEFB genes and for AK125948 gene.

Supplementary Table S3 shows the pairwise LD (D') for the 17 SNPs genotyped in the whole family set.

Supplementary Table S4 shows the results for HLA-Cw6 tagging SNPs (PSORSl locus).

Supplementary Table S5 shows the results for most significant under- and over- transmitted ADAM33 3-SNP haplotypes when stratifying according to HLA-Cw6 status in patients (for Set I)

Description of the Preferred embodiments

The present invention is based on the discovery by the present inventors that particular single nucleotide polymorphisms (SNPs) of the ADAM33, and potentially ADAM33 alpha and/or beta iso forms expression, are correlated with psoriasis.

Thus, in a first aspect, the present invention provides a method of testing a subject thought to have or be predisposed to having psoriasis which comprises the step of analyzing a biological sample from said subject for :

(i) detecting the presence of a SNP in the ADAM33 gene, which SNP is associated with psoriasis , and/or (ii) analyzing the expression of the ADAM33 gene.

Said method, by detecting the presence of a SNP in the ADAM33 gene associated with psoriasis or by determining the expression of the ADAM33 gene in a subject enables to confirm that said subject has or is predisposed for having psoriasis.

As used herein, the term "subject" refers to a mammal, preferably a human.

As used herein, the expression "biological sample" refers to solid tissues such as, for example, a lung biopsy; buccal swab, fluids and excretions such as for example, sputum, induced sputum, blood, serum, plasma, urine. Preferably, said biological sample is a fluid sample and most preferably a blood sample.

As used herein, the expression "ADAM33 gene" refers to the ADAM metallopeptidase domain 33 which is well known from one of skill in the art. As an example, and for Homo sapiens, the ADAM33 alpha isoform mRNAs has the sequence SEQ ID NO:1 (NM 025220) or the sequence SEQ ID NO:2 for ADAM33 beta isoform mRNA 2 (NM_153202), the ADAM33 preproprotein alpha isoform has the sequence SEQ ID NO: 3 (NP 079496) or the ADAM33 preproprotein beta isoform has the sequence SEQ ID NO:4 for variant 2 (NP_694882), and the ADAM33 gene has the sequence SEQ ID NO:5 (NC 000020).

Preferably, the expression of the ADAM33 gene refers to the expression of the alpha and/or beta of the ADAM33 gene.

In view of the examples, the skilled person will be able to identify the SNPs in the

ADAM33 gene associated with psoriasis. As an example, one can cites rs512625 (nucleotide N at position 31 of SEQ ID NO:6, wherein allele A is associated to psoriasis), rs677044 (nucleotide N at position 31 of SEQ ID NO:7, wherein allele G is associated to psoriasis), rs597980 (nucleotide N at position 31 of SEQ ID NO:8, wherein allele T is associated to psoriasis), rs44707 (nucleotide N at position 31 of SEQ ID NO:9, wherein allele C is associated to psoriasis), rs628977 (nucleotide N at position 31 of SEQ ID NO:21, wherein allele T is associated to psoriasis), rs598418 (nucleotide N at position 27 of SEQ ID NO:22, wherein allele C is associated to psoriasis), rs2853209 (nucleotide N at position 27 of SEQ ID NO:23, wherein allele A is associated to psoriasis), rs2787095 (nucleotide N at position 27 of SEQ ID NO:24, wherein allele C is associated to psoriasis), rs2853213 (nucleotide N at position 27 of SEQ ID NO:25, wherein allele G is associated to psoriasis), rs 1046919 (nucleotide N at position 27 of SEQ ID NO:26, wherein allele C is associated to psoriasis).

Preferably, said SNPs are selected in the group comprising or consisting of rs512625 (nucleotide N at position 31 of SEQ ID NO:6, wherein allele A is associated to psoriasis), rs677044 (nucleotide N at position 31 of SEQ ID NO:7, wherein allele G is associated to psoriasis), rs597980 (nucleotide N at position 31 of SEQ ID NO:8, wherein allele T is associated to psoriasis), rs44707 (nucleotide N at position 31 of SEQ ID NO:9, wherein allele C is associated to psoriasis)

The skilled man will immediately appreciate that the information presented herein relating to the human ADAM33 may easily be equated or correlated with a similar mutation at a corresponding location for the ADAM33 gene from another species.

Typical techniques for detecting single nucleotide polymorphisms may include dynamic allele-specific hybridisation, ligation chain reaction, mini-sequencing, DNA"chips", allele-specific oligonucleotide hybridisation with single or dual-labelled probes merged with PCR or with molecular beacons, and others.

Analyzing the expression of the ADAM33 gene may be assessed by any of a wide variety of well-known methods for detecting expression of a transcribed nucleic acid or translated protein.

In a preferred embodiment, the expression of ADAM33 gene is assessed by analyzing the expression of ADAM33 alpha and/or beta mRNA transcripts or mRNA precursors, such as nascent RNA, of said gene. Said analysis can be assessed by preparing mRNA/cDNA from cells in a biological sample from a subject, and hybridizing the mRNA/cDNA with a reference polynucleotide. The prepared mRNA/cDNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses, such as quantitative PCR (TaqMan), and probes arrays such as GeneChip™ DNA Arrays (AFFYMETRIX). Advantageously, the analysis of the expression level of mRNA transcribed from ADAM33 gene involves the process of nucleic acid amplification, e. g., by RT-PCR (the experimental embodiment set forth in U. S. Patent No. 4,683, 202), ligase chain reaction (BARANY, Proc. Natl. Acad. Sci. USA, vol.88, p: 189-193, 1991), self sustained sequence replication (GUATELLI et al., Proc. Natl. Acad. Sci. USA, vol.87, p: 1874-1878, 1990), transcriptional amplification system (KWOH et al., 1989, Proc. Natl. Acad. Sci. USA, vol.86, p: 1173-1177, 1989), Q-Beta Replicase (LIZARDI et al., Biol. Technology, vol.6, p: 1197, 1988), rolling circle replication (U. S. Patent No. 5,854, 033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3 'regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.

In another preferred embodiment, the expression of ADAM33 gene is assessed by analyzing the expression of the protein translated from said gene. Said analysis can be assessed using an antibody (e.g., a radio-labeled, chromophore-labeled, fluorophore- labeled, or enzyme-labeled antibody), an antibody derivative (e.g., an antibody conjugate with a substrate or with the protein or ligand of a protein of a protein/ligand pair (e.g., biotin-streptavidin)), or an antibody fragment (e.g., a single-chain antibody, an isolated antibody hypervariable domain, etc.) which binds specifically to the protein translated from ADAM33 gene.

Said analysis can be assessed by a variety of techniques well known by one of skill in the art including, but not limited to, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis and enzyme linked immunoabsorbant assay (ELISA).

Polyclonal antibodies against ADAM 33 can be prepared by immunizing a suitable animal, such as mouse, rabbit or goat, with said protein. The antibody titer in the immunized animal can be monitored over time by standard techniques, such as with an ELISA using immobilized polypeptide. At an appropriate time after immunization, e.g., when the specific antibody titers are highest, antibody producing cells can be obtained from the animal and used to prepare monoclonal antibodies (mAb) by standard techniques, such as the hybridoma technique originally described by KOHLER and MILSTEIN (Nature, vol.256, p:495-497, 1975), the human B cell hybridoma technique (KOZBOR et al., Immunol, vol.4, p: 72, 1983), the EBV- hybridoma technique (COLE et al., In Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc, p: 77-96, 1985) or trioma techniques. The technology for producing hybridomas is well known (see generally Current Protocols in Immunology, COLIGAN et al. ed. , John Wiley & Sons, New York, 1994). Hybridoma cells producing the desired monoclonal antibody are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide of interest, e.g., using a standard ELISA. Finally, antibodies against ADAM33 can be already obtained from ABCAM, ABR- Affinity BIOREAGENTS, GENETEX, NOVUS BIOLOGICALS, or from SANTA CRUZ BIOTECHNOLOGY, INC.

The method of the invention may comprise comparing the level of expression of ADAM33 gene in a biological sample from a subject with the normal expression level of said gene in a control.

According to a preferred embodiment, a significantly higher level of expression of said gene in the biological sample of a subject as compared to the normal expression level is an indication that the patient has or is predisposed to psoriasis.

A "higher level of expression" of ADAM33 gene, preferably from ADAM33 alpha and/or beta isoforms, refers to an expression level in a biological sample that is greater than the standard error of the assay employed to assess expression, and is preferably at least 20% superior to the normal level of expression of said gene, preferably at least 50% superior to the normal level of expression of said gene, and most preferably at least 100% superior to the normal level of expression of said gene.

According to another preferred embodiment, a significantly lower level of expression of said gene in the biological sample of a subject as compared to the normal expression level is an indication that the patient has or is predisposed to psoriasis.

A "lower level of expression" of ADAM33 gene, preferably from ADAM33 alpha and/or beta isoforms, refers to an expression level in a biological sample that is lower than the standard error of the assay employed to assess expression, and is preferably at least 20% inferior to the normal level of expression of said gene, preferably at least 30% inferior to the normal level of expression of said gene, and most preferably at least 50% inferior to the normal level of expression of said gene.

The "normal" level of expression of ADAM33 gene is the level of expression of said gene in a biological sample of a subject not afflicted with psoriasis. Preferably, said normal level of expression is assessed in a control sample (e.g., sample from a healthy subject, which is not afflicted by psoriasis) and preferably, the average expression level of said gene in several control samples.

In another aspect, the present invention provides a use, for treating and/or preventing psoriasis in a subject, of a compound which specifically inhibits the expression of ADAM33 gene, preferably from ADAM33 alpha and/or beta isoforms.

Preferably, said compound specifically inhibiting the expression ADAM33 gene is an oligonucleotide, which is selected from the group comprising anti-sense RNA and DNA molecules, ribozymes, siRNAs and aptamers.

More specifically, anti-sense RNA and DNA molecules and ribozymes function to inhibit the translation of ADAM33 mRNA. Anti-sense RNA and DNA molecules act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation. With regard to anti-sense DNA, oligodeoxyribonucleotides derived from the translation initiation site, e. g. , between - 10 and +10 regions of the ADAM33 nucleotide sequence, are preferred. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by a endonucleo lytic cleavage. Within the scope of the invention are engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleo lytic cleavage of ADAM33 RNA sequences. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable. Both anti-sense RNA and DNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the anti- sense RNA molecule.

Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, anti-sense cDNA constructs that synthesize anti-sense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

Short interference RNA molecules (siRNA) can also be used for inhibiting the expression of ADAM33 gene. Said interference RNA molecules can be generated based on the genetic sequences of ADAM33 gene. RNA interference (RNAi) is based on the degradation of particular target sequences by the design of short interference RNA oligo's (siRNA) which recognize the target sequence (here ADAM33) and subsequently trigger their degradation by a poorly understood pathway. In general siRNA duplexes are shorter than 30 nucleotides, because longer stretches of dsRNA activate the PKR pathway in mammalian cells which results in a global a-specific shut-down of protein synthesis. Preferably, the length of said siRNA is comprised between 15 and 25 bp

(bases pair), and most preferably between 19 and 24 bp. The preparation and gene therapy vectors for the intracellular expression of siRNAs duplexes is disclosed in W00244321. As an example of siRNA, one can cite SEQ ID NO: 10

(GGUUCUGGAGCUCAGAAUC), SEQ ID NO: 11 (GCAGAUCAAGUCCAGAUGC), SEQ ID NO: 12 (GUUCAACACUGCAGUGAGC), SEQ ID NO: 13 (AGACAUGUUGGCUAUAGGC), SEQ ID NO: 14 (GAGCCACAUUAGAAGUUCC), SEQ ID NO: 15 (UAGCAACCAUAACUGCCAC), SEQ ID NO: 16 (AACCAUGACACCUUCCUGC), SEQ ID NO: 17 (GACAUGUUGGCUAUAGGCG), SEQ ID NO: 18 (ACCAUAACUGCCACUGUGC), SEQ ID NO: 19 (AGUCCAGAUGCCAAGAUCC), and SEQ ID NO: 20 (ACUGUACAUUGUGGCAGAC).

RNA aptamers can also be used for inhibiting the expression of ADAM33 gene.

Recently, RNA aptamers have been used as therapeutic reagents for their ability to disrupt protein function. Selection of aptamers in vitro allows rapid isolation of extremely rare RNAs that have high specificity and affinity for specific proteins. Exemplary RNA aptamers are described in U. S. Pat. No. 5,270,163, in GOLD et al. (Nature, vol.346, p:818-822, 1990), and TUERK & GOLD (Science, vol.249, p:505- 510, 1990). Unlike anti-sense compounds, whose targets are one dimensional lattices, RNA aptamers can bind to the three dimensional surfaces of a protein. Moreover, RNA aptamers can frequently discriminate finely among discrete functional sites of a protein (GOLD et al, Annu. Rev. Biochem., vol.64, p:763-797, 1995). As research and therapeutic reagents, aptamers not only have the combined advantages of antibodies and small molecular mass drugs, but in vivo production of RNA aptamers also can be controlled genetically. Such RNA expressing genes are usually smaller than protein- coding genes and can be inserted easily into gene therapy vectors.

Preferably, said oligonucleotide is a siRNA.

The oligonucleotide may be delivered in vivo alone or in association with a vector.

In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the siRNA to the cells.

As an example, such oligonucleotide can be associated with non-lipid cationic polymers (WU and WU, J. Biol. Chem., vol.263, p: 14621-4, 1988) or liposomes (BRIGHMAN et al, Am. J. Med. ScL, vol.298, p: 278-81, 1989) to form complexes enhancing cellular uptake.

In still another aspect, the present invention provides a use, for treating and/or preventing psoriasis in a subject, of a compound which specifically increase the expression of ADAM33 gene, preferably from ADAM33 alpha and/or beta isoforms.

Preferably, said compound specifically increasing the expression ADAM33 gene is an oligonucleotide expression vector coding for the ADAM33 protein, which vector may be for example in the form of a plasmid, a viral particle, a phage, etc.

Such vectors may include bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. Large numbers of suitable vectors are known to those of skill in the art and are commercially available.

The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (QIAGEN), pbs, pDIO, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNHlβa, pNH18A, pNH46A (STRATAGENE), ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (PHARMACIA). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTl, pSG (STRATAGENE), pSVK3, pBPV, pMSG, pSVL (PHARMACIA). However, any other vector may be used as long as it is replicable and viable in the host.

The polynucleotide sequence, preferably the DNA sequence in the expression vector coding for ADAM33 protein is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, one can mentioned prokaryotic or eukaryotic promoters such as

CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. The expression vector also contains a ribosome binding site for translation initiation and a transcription vector. The vector may also include appropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli. The vector of the invention containing the appropriate polynucleotide sequence as herein above described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the polypeptide.

As an example, transcription of a DNA encoding for the polypeptide described previously by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. Enhancer are cis-acting elements of DNA, usually about from 10 to 300 pb that act on a promoter to increase its transcription. Examples of enhancer include the

SV40 enhancer, the CMV early promoter enhancer, and adenovirus enhancers.

Such oligonucleotide expression vector can be associated with non-lipid cationic polymers (WU and WU, J. Biol. Chem., vol.263, p: 14621-4, 1988) or liposomes (BRIGHMAN et al, Am. J. Med. ScL, vol.298, p: 278-81, 1989) to form complexes enhancing cellular uptake.

An effective amount of compound is one which is sufficient to achieve a desired biological effect, in this case inducing an increased or decreased expression of ADAM33 gene, preferably from ADAM33 alpha and/or beta iso forms. It is understood that the effective dosage will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. The ranges of effective doses provided below are not intended to limit the invention and represent preferred dose ranges. However, the preferred dosage can be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation.

Advantageously, said compound specifically inhibiting or increasing the expression ADAM33 gene may be associated with a pharmaceutically acceptable vehicle.

As an example of pharmaceutically acceptable vehicle, the composition may comprise emulsions, microemulsions, oil-in-water emulsions, anhydrous lipids and oil- in-water emulsions, other types of emulsions. The composition may also comprise one or more additives (e.g., diluents, excipients, stabilizers, preservatives). See, generally, Ullmann 's Encyclopedia of Industrial Chemistry, 6th Ed. (various editors, 1989-1998, Marcel Dekker); and Pharmaceutical Dosage Forms and Drug Delivery Systems (ANSEL et al, 1994, WILLIAMS & WILKINS).

In still another aspect, the present invention provides an in vitro method of selecting a compound, which can be useful for treating psoriasis, characterized in that said method comprises the steps of:

a) obtaining a cell expressing ADAM33 gene;

b) contacting said cell with at least one compound,

d) selecting the compound, which induces a lower level of expression of the

ADAM33 gene in the cell contacted to that compound.

a) obtaining a cell expressing AD AM33 gene;

b) contacting said cell with at least one compound,

As used herein, the term "compound" refers to any type of molecules such as polypeptides, polynucleotides, sugars, lipids, or any other chemical compounds.

Methods for determining the expression of the ADAM33 are well known from one of skill in the art. As an example, one can cite the methods which have been described previously. In the following, the invention is described in more detail with reference to nucleic acid sequences and the examples. Yet, no limitation of the invention is intended by the details of the examples. Rather, the invention pertains to any embodiment, which comprises details which are not explicitly mentioned in the examples herein, but which the skilled person finds without undue effort.

EXAMPLES

PATIENTS, MATERIALS AND METHODS

Families: The French psoriasis study was approved by the Ethics Committee of Le Kremlin-Bicetre Hospital in 1995 (CCPPRB) [MAHE et al, Eur. J. Dermatol, vol.12, p:66-69, 2002]. Briefly, families were identified through a media campaign between 1996 and 2001 at Genethon, using posters in the Paris Metro and information in news magazines, radio and television. Clinical diagnoses were checked by systematic telephone calls to every family member, affected or non-affected, for each family, at least twice during four years by dermatologists using a standard questionnaire. The attending physician of each patient was also contacted, usually by mail, which led to confirmation of the diagnosis in over 75% of cases. Thus, 126 families were enrolled in the genetic study and provided blood samples. These families were divided in two non- homogeneous family sets (Set I and Set II): Set I corresponded to the 45 highly predisposed multigenerational families used for the initial genome-wide scan, and included on average 8 affected members per family [LESUEUR et al, abovementionned, 2007], whereas Set II corresponded to the 81 remaining smaller families (3 affected members per family on average, Table 3). One inbred large family was reported in Set I, with parents who were first cousins (inbred coefficient for children = 1/16). DNA was extracted from whole blood using standard procedures after written informed consent of subjects. The study was conducted in accordance with the Declaration of Helsinki Principles.

SNP selection: SNPs were initially identified through the HapMap database. A list of 402 validated SNPs located between the microsatellite markers D20S864 and

D20S112 was generated. In order to perform a first scanning of the region with a limited number of SNPs, biallelic markers were filtered according to the following criteria: selected markers had a minor allele frequency (MAF) > 20% in Caucasian populations, were located within known genes or nearby (+10 kb upstream and downstream of known genes), and SNPs with ambiguous flanking sequences were excluded for genotyping. The population frequencies for the SNPs were taken from the CEU HapMap population (CEPH collection of Utah residents of northern and western European ancestry). Thus, 85 SNPs whose position was representative of the overall marker distribution were eligible for genotyping for Stage I. These SNPs were located in or near 65 different genes and were not in linkage disequilibrium with each other (1 SNP/ 137 kb on average).

To select additional SNPs in the 3 candidate regions on 2Op 13 (Stage II), different strategies were applied depending on the availability of SNP data for each candidate gene. When coverage of a gene with HapMap SNPs was sufficient, we used the Tagger program to select SNPs that efficiently tagged all common variations of the candidate genes [DE BAKKER et al, Nat. Genet., vol.37, p: 1217-1223, 2005]. This was the case for SIGLECl and AKl 25945: 17 HapMap tagging SNPs across SIGLECl (Table 1) and five HapMap tagging SNPs across AKl 25945 (Supplementary Table S2) were genotyped in family set I. For these two genes, two additional common SNPs that were absent in HapMap database (rslO18493, located in exon 6 of SIGLECl and rsl060236, located in the untranslated region of AKl 25945) were also genotyped.

When coverage with HapMap SNPs was insufficient, the density of markers across a candidate was increased using validated SNPs from dbSNP database or from the literature. This was the case for ADAM33. This gene had been resequenced in different populations [VAN EERDEWEGH et al, Nature, vol.418, p:426-430, 2002;

CHAE et al., J. Hum. Genet., vol.48, p:278-281, 2003], and a number of reported SNPs were not present in the HapMap database. Therefore, in addition to 8 HapMap SNPs, 15 other SNPs were selected. Those included all 3 validated nonsynonymous SNPs (with frequency > 5% in Caucasians) and SNPs that had been previously shown to be associated with asthma either in univariate or in haplotype analyses. These SNPs included F_-l (rs3918392), F_+l (rs511898), Q_-l (rs612709), S_l (rs3918396),

ST_+4 (rs44707), ST_+7 (rs574174), V_-2 (rs628977), V_-l (rs543749) and V_4 (rs2787094) [VAN EERDEWEGH et al, abovementionned, 2002] (Table 1). Two additional SNPs previously found to be associated with asthma, V_2 (rs3918400) and V_5 (rs3746631), did not assay successfully; however, given the moderate to strong levels of LD of SNPs previously documented, we expected the studied SNPs across the region to capture most of the haplotypic variation.

Very few SNPs had been identified in GFRA4, despite the fact that the whole coding sequence of the gene had been resequenced ([CEBRIAN et al., J. Clin. Endocrinol. Metab., vol.90, p:6268-6274, 2005] and personal communication). Therefore, only 4 SNPs were selected for genotyping in our family set.

Finally for genes within Region 1 (DEFB 125, DEFB 126, DEFBl 27, DEFBl 28, DEFB 129 and DEFB 32), incomplete SNP data were available in the public SNP databases when this study was initiated. Therefore, we resequenced the entire coding sequence and the exon/intron junctions of the 6 DEFB genes in 58 unrelated Caucasian controls. Tagging SNPs were identified using the tagsnps program [STRAM et al., Genet. Epidemiol., vol.27, vol:365-374, 2004].

SNP genotyping: Genotyping was carried out using Taqman^® according to manufacturer's instructions. Primers and probes were supplied directly by Applied

Biosystems as Assays-by-Design™. All assays were carried out in 384-well plates.

Each plate included negative controls (with no DNA) and positive controls were duplicated on a separate quality control plate. Plates were read on the ABI PRISM 7900 using the Sequence Detection Software (Applied Biosystems, Foster City, California, United States). Failed genotypes were not repeated.

Genotypes were checked for Mendelian inheritance errors using FBAT [LANG & LAIRD, abovementionned, 2002] and PEDSTATS was used to discard SNPs which deviate from Hardy- Weinberg Equilibrium in unrelated subjects [WIGGINTON & ABECASIS, Bioinformatics, vol.21, p:3445-3447, 2005].

Statistical analyses: Family based association analysis was carried out using the

FBAT program to examine the transmission rates of marker alleles under the assumption of linkage. The FBAT test is a multiallelic test based on the classic transmission/disequilibrium test (TDT) developed by SPIELMAN et al. [Am. J. Hum. Genet., vol.52, p:506-516, 1993]. It considers parents heterozygous for a certain allele at the marker locus associated with the disease and evaluates the frequency with which that allele is transmitted to affected offspring. In each trio, the untransmitted alleles of the parents serve as controls. The FBAT method permits analysis of family structures larger than trios. It has been shown that, when data on parents are missing, one case and two sibs bring similar power levels to trios and adding sibs when parents are available increases power [LANG & LAIRD, abovementionned, 2002]. The FBAT software decomposes pedigrees into individual nuclear families and treats them as independent in most of the calculations. The pedigree's contribution to the FBAT test statistics is then obtained by summing over all nuclear families within the pedigree. However, in the case where linkage is present and the null hypothesis states "linkage, but no association", the genotypes of the different nuclear families derived from one pedigree are correlated. Even with a single nuclear family, the transmissions to multiple sibs are correlated when linkage is present. Therefore, when testing for association in an area of known linkage with multiple sibs in a family or when multiple families in pedigree occur, an empirical variance for the test statistics should be used. We used the -e option of FBAT to compute the "corrected" test statistic, and gave the P-value "P" corresponding to this corrected test statistic. Furthermore, haplotype analysis was performed using the HBAT function of FBAT, under the assumption of linkage. This is a method for estimating genetic association from probabilities of haplotype transmission to affected offspring. To circumvent the problem of multiple testing due to the large number of statistical tests performed simultaneously in the association study, the false discovering rate was controlled and permutation P-values were computed with FBAT program (1,000,000 permutation tests were performed).

Electronic databases and programs:

HapMap data are available at "http://www.hapmap.org" (public data released n° 6 at 2005-03-01).

The dbSNP database is available at "http://www.ncbi.nlm.nih.gov/SNP/".

The University of California Santa Cruz assembly of the genome is available at ' 'http : //genome .ucsc . edu/' ' . The FBAT program version 1.5.5 is available at "http://www.biostat.harvard.edu/~fbat/".

PEDSTATS is available at "http://www.sph.umich.edu/csg/abecasis/PedStats/".

Tagsnps is available at "http://www-rcf.usc.edu/~stram/tagSNPs.html".

Tagger on Haploview, version 3.2 is available at

"http://www.broad.mit.edu/mpg/haploview/using.php".

RESULTS

Stage I: Preliminary screen for SNP-based association on Chromosome 2Op 13

The figure IA shows a genetic map on Chromosome 2Op 13 of a linkage interval associated with psoriasis (tel, telomere; cen, centromere). Position of microsatellites used in the linkage analysis is indicated in centimorgans (cM).

The 17Mb candidate locus on 2Op 13 extended from the telomere of the short arm of Chromosome 20 (D20S864) to the microsatellite D20S112 (Figure IA), and contains 428 known genes. We aimed to define the boundaries of this region of linkage and to identify the causative variants. We initially selected 85 SNPs across the region for genotyping. For all SNPs, genotypes in founders satisfied the Hardy-Weinberg equilibrium.

Figure IB illustrates the results of the family-based association test (FBAT) under the assumption of linkage [LANG & LAIRD, Am. J. Hum. Genet., vol.71, p:575-584, 2002] for the 85 SNPs genotyped in 45 multigenerational families (corresponding to 295 nuclear families).

The figure IB shows the Z plot for association tests performed with FBAT under the assumption of linkage, for SNPs selected in Stage I. Position of SNPs is indicated in megabases (Mb). The dotted line indicates the threshold for significance of the association test (a Z score > 3 corresponds to a P < 0.05). Position, minor allele frequency, and FBAT results for all SNPs are given in Supplementary Table S 1. The figure 1C. shows the detailed physical map on the contig NT Ol 1387.8 at the 3 candidate loci. Positions of the SNPs genotyped in stage I and in stage II are shown with arrows. Red arrows indicate SNPs showing evidence for association (P < 0.05) in stage I; orange arrows indicate SNPs showing evidence for association (P < 0.05) in stage II; black arrows indicate SNPs showing no evidence for association in the univariate analysis.

Four SNPs showed some evidence for association to psoriasis (P < 0.05): rsl2480529 located in the promoter of DEFB 127, rsβl 10460 located in intron 1 of DEFB129, rs512625 located in the 3'UTR of ADAM33, and rs6053417 located in the promoter of AKl 25948 encoding a hypothetical protein (Figure 1C).

Stage II: Haplotype analyses of candidate genes at the 3 loci

We used data from the International HapMap project to look at patterns of LD surrounding the SNPs showing some association with psoriasis in the CEPH Caucasian sample set [The International Psoriasis Genetics Study, abovementionned, 2003]. The Defensin B (DEFB) gene cluster, ADAM33 and AKl 25948 were located in 3 distinct regions (Figure 1C) and the associated markers were not in LD. To capture most of the genetic variation at the three loci, and to potentially identify allelic variant(s) predisposing to psoriasis, a tagging SNP approach was undertaken to test the candidate genes in the 3 regions (see Patients, Materials and Methods section for SNP selection). A total of 63 additional SNPs were genotyped in the same family set. Sixteen SNPs were genotyped within Region 1 , 45 SNPs within Region 2 and 6 SNPs within Region 3 (Figure 1C). There was no evidence for association between the DEFB genes and psoriasis, nor between AKl 25948 and psoriasis in the univariate SNP analyses and in the haplotypes analyses (Supplementary Table S2). However, association was observed for four additional SNPs genotyped within Region 2: three of them were ADAM33 intronic SNPs (rs677044, rs597980, rs44707), and the fourth one (rs6076542) was in intron 3 of SIGLECl, a gene located 5kb from ADAM 33. Results of the association tests for the 45 SNPs in Region 2 are presented in Table 1.

Evidence for effects of combinations of ADAM ^'3 '3 SNPs on the risk of psoriasis Due to the low pairwise LD (Supplementary Table S3) and to the elevated number of haplotypes generated at the ADAM33 locus, haplotypes covering the whole region could not be constructed. However, because multiple SNPs may act in combination to alter the risk of psoriasis, transmissions of all possible 2- or 3-SNP haplotypes to affected individuals were successively examined. The best association for the 2-SNP haplotypes was obtained for the pair SNP5/SNP23 (P=0.0005, based on 1,000,000 permutations). But generally stronger associations were obtained for the 3-SNP haplotypes. The ten best 3-SNP haplotypes are presented in Table 2. Associated haplotypes involved exclusively SNPs within ADAM33, and revealed both risk and protective effects of ADAM33 alleles depending on the SNP combination.

Association of ADAM33 with psoriasis is independent of HLA-Cw6 status

The 2Op 13 locus is likely to segregate independently of PSORSl in the psoriasis families [LESUEUR et ah, abovementionned, 2007]. However, a potential interaction between ADAM33 and HLA-Cwβ alleles was tested. HLA-Cw6 positive patients were identified using SNPs in strong LD with the risk allele (namely HCR-325 C>T (rsl30076), HCR-1723 G>T (rsl30079), HCR-2327 OG (rsl576) and CDSN971 OT (rsl062470) [24,25]. As expected, those markers define a strongly associated haplotype in our population (P<0.000001 for haplotype H2, based on 1,000,000 permutations, Supplementary Table S4). Association between ADAM 33 and psoriasis was then monitored using FBAT when stratifying the families according to the presence or absence of this risk haplotype. Although the number of informative families was reduced, the associations between ADAM33 3-SNPs haplotypes and psoriasis were still observed in the group of patients not carrying HLA-Cw6, indicating that the 2 loci act independently. As expected, the associations were less significant in the group of patients carrying HLA-Cw6, due to the stronger contribution of the 6p21 locus to psoriasis susceptibility (Supplementary Table S5).

Genetic heterogeneity between large, multigenerational families and smaller families segregating psoriasis

We attempted to confirm our findings of association between ADAM33 and psoriasis in a broader family sample. Thus, a second set of 81 smaller French families was investigated (Set II). Those families had not been included in the linkage study due to the low informativity of the pedigrees for such studies. However, they represented 173 nuclear families on which the FBAT transmission disequilibrium test could be performed (Table 3). Therefore, the 15 SNPs contributing to the best combinations were genotyped in Set II (Table 4). No association was found in the individual or combined SNP analyses when analyzing Set II independently, but same 3-SNP haplotype associations were confirmed when combining Set I and Set II. It is likely that the discrepancy between the 2 family sets was due to a lower informativity of Set II because of the difference in the pedigree structures (Table 3). Indeed, the numbers of informative nuclear families for the 3-SNP haplotypes giving the best associations in Set I were lower in Set II than in Set I (Table 2). Moreover, the results of HLA-C tagging SNPs, known to be strongly associated with psoriasis, illustrated the weak power of Set II for performing family-based association tests. For example, for SNP rsl062470, a P of 0.0001 was obtained for Set I, which had 86.7% (39/45) of informative families, whereas a P of 0.02 was obtained for Set II but this set had only 50.6% (41/81) of informative families. However, a significant association was observed for the combined sets (P=O.000006, Supplementary Table S4).

DISCUSSION

A common genetic component to autoimmune susceptibility had been initially shown by twin and adoption studies and by increased risk to siblings [VYSE & TODD,

Cell, vol.85, p:311-318, 1996]. Later, linkage studies demonstrated that autoimmune diseases share a limited number of loci [BECKER et ah, Proc. Natl. Acad. Sci. U.S.A, vol.95, p:9979-9984, 1998; COOKSON, Nature, vol.402, p:B5-l l, 1999], reinforcing the idea that common susceptibility genes control them. Here, using a family-based association design to interrogate the locus, we identified several combinations of SNPs within ADAM33, a gene that has been associated with asthma in many studies, to be strongly associated with psoriasis in multigenerational French families.

Due to the large number of all possible 2- and 3-SNP combinations tested, the issue of multiple testing should be addressed here. A Bonferroni correction, although too conservative because the SNPs in the candidate region were all correlated, could be applied. When considering all possible 2-SNP combinations with one of the 5 SNPs associated in the univariate analysis, 110 tests are performed. Therefore, the threshold for a significant P- value should be <0.00045. The best 2-SNP combination (SNP5/SNP23) gives a P=0.0005, based on 1,000,000 permutations, in favour of a significant association with psoriasis. Moreover, these 2 SNPs are also included in some of the best 3-SNP combinations (Table X), which is again in favor of a true association.

These associations were not observed in a set of much smaller families. Although statistical type I errors in Set I cannot be totally discarded here, the discrepancy between the 2 family sets could be accounted for by a lack of informativity of Set II families. Another possible explanation could be that the selection of highly predisposed family enriched Set I for individuals carrying risk alleles at a smaller number of loci with stronger effects, whereas psoriasis susceptibility in Set II may be due to a higher number of loci with weaker effects. Thus, the contribution of specific ADAM33 alleles to the familial clustering and individual risk prediction of psoriasis is likely to be relatively small, and these issues of case selection should be addressed in future replication studies.

Nevertheless, our findings can be the source of valuable physiological insights, since several ADAM33 SNPs have been found to be associated with asthma and with bronchial hyper-responsiveness in Caucasians, in African Americans and in Hispanics [VAN EERDEWEGH et al, abovementionned, 2002; HOWARD et al, J. Allergy Clin. Immunol, vol.112, p:717-722, 2003 ; WERNER et al, Clin. Exp. Allergy, vol.34, p:26- 31, 2004]. An association of ADAM33 with allergic rhinitis has also been reported in the Japanese population [CHENG et al, Clin. Exp. Allergy, vol.34, p:l 192-1201, 2004]. This first report of an association between ADAM33 and psoriasis confirms that common biological pathways may be involved in the etiology of psoriasis and other clinically distinct immune-mediated diseases.

Although clinical data on inflammatory and autoimmune diseases other than psoriasis were limited in our family sample, we examined personal or family history of atopy (AD, asthma and allergic rhinitis) and of seborrheic dermatitis (SD) retrospectively from the data available from the questionnaire answered by family members (Set I). SD was reported in 34 families (75.6%, 128 subjects), AD in 25 families (55.6%, 63 subjects), asthma in 22 families (47.8%, 34 subjects), and allergic rhinitis in 5 families (10.9%, 5 subjects). These data indicate a higher incidence of chronic inflammatory diseases in the psoriasis families than in the general population, except for allergic rhinitis (Incidences of SD, AD, asthma and allergic rhinitis in France are respectively: 1-3%, 2-5%, 5-7% and 15%) and support the existence of common genes interacting with other genetic or environmental factors to result in distinct immunologic abnormalities. As in the general population, these diseases rarely occurred in the same patient in our family sample, indicating that susceptibility alleles for these disorders are likely to be different. Indeed, ADAM33 SNPs that have been associated with asthma are not the SNPs defining protective and risk haplotypes for psoriasis [VAN EERDEWEGH et al, abovementionned, 2002]. The recently reported colocalization of the susceptibility loci for psoriasis (PSORS4) and atopic dermatitis (ATOD2) on Chromosome Iq21 also supports this hypothesis [GIARDINA et al, Hum. Hered., vol.61, p:229-236, 2006]. Although immunologic processes in psoriasis and AD are quite different and the two diseases rarely occur together in the same patient, the possibility of a specific misregulation of the LOR gene at Iq21, which is down- regulated in psoriasis, and up-regulated in AD has been suggested [GIARDINA et al, abovementionned, 2006]. In the light of our data, the involvement oϊ AD AM 33 should be further investigated in AD as well.

Once an allelic association with the disease has been demonstrated, the identification of causal variants is less straightforward. The ADAM33 gene consists of

22 exons that have been re-sequenced in different populations for SNP identification

[VAN EERDEWEGH et al, abovementionned, 2002, CHAE et al, abovementionned,

2003]. Of the numerous SNPs described in the public SNP databases, only 4 validated

SNPs occur in the coding region of the gene and, of these, 3 are non- synonymous. We have excluded an association between two of them, T764M (rs2280091) and S774P

(rs2280090), and psoriasis (Table 1). The third SNP, A178T (rs3918392), was also genotyped in our family set, but association tests could not be performed due to its low frequency (3%) in our population. In asthma studies, it has been proposed that 3'UTR polymorphisms may be significant [HOLGATE et al, Thorax, vol.58, p:466-469, 2003], although functional investigations of some of them have so far been unsuccessful

[UMLAND et al, Am. J. Respir. Cell. MoI Biol, vol.29, p:571-582, 2003].

Interestingly, SNP 7 (rs677044) in the 3'UTR of ADAM33 showed some association when analyzed on its own and was present on all 4 most significant protective haplotypes (Table 2). However, this SNP was also present on other haplotypes not associated with the disease. Therefore, a functional role of this SNP in psoriasis susceptibility should be discarded. Finally, ADAM33 gene undergoes complex alternative splicing with several variant transcripts and their relative functional significance to each other is not clear [UMLAND et al, abovementioned, 2003, POWELL et al, Am. J. Respir. Cell. MoI Biol, vol.31, p:13-21, 2004]. It has been suggested that some of the ADAM33 polymorphisms may affect alternative splicing, splicing efficiency or mRNA turnover [VAN EERDEWEGH et al, abovementionned, 2002] but such functional effects for SNP5 (rs512625) in the 3' region OΪADAM33 and for SNP14 (rs597980) and SNP15 (rs44707) in intron 19 of the gene were not investigated in this study.

It has already been noticed that the individual effect of a variant can be too weak to be detected individually and that interactions of multiple SNPs within the same gene can affect a phenotype [DRYSDALE et al, Proc. Natl Acad. ScL U.S.A, vol.97, p: 10483-10488, 2000]. In complex situations of gene involvement, JANNOT et al showed that testing combinations of SNPs could provide better power than testing each single SNP for association [Genet. Epidemiol, vol.25, p: 158-167, 2003]. In type 2 diabetes, CAPNlO and NOD2 are two examples where haplotypes made up of non- coding variants have been associated with disease phenotypes in complex fashion while no association was seen in the univariate SNP analyses [COX et al, Diabetes, vol.53, p:S19-25, 2004]. The same situation is observed in the case of asthma and psoriasis, where the association with ADAM33 is stronger when combinations of SNPs are examined.

A recent study confirmed that the ADAM33 locus shows extended linkage disequilibrium upstream of ADAM33 to GFRA4, as well as downstream including SIGLECl (also named sialoadhesin SN) [WJST, Allergy, vol.62, p:444-446, 2007]. The region can be divided into 5 haplotype blocks, ADAM33 being situated between block 4 and 5, with an increased recombinatory rates around exons S to V of ADAM 33. Half of the SNPs included in the associated combinations here lie in exon S or upstream (SNPs 5, 7, 9, 10, 11, 15, 16) and the second half lie downstream exon F (SNP 21, 23, 24, 25, 26, 27). Resolution of LD maps and block definition at ADAM33 locus is still noisy and it is likely that yet unidentified variant(s) within the ADAM33 gene or within distant regulatory elements may be responsible for asthma or psoriasis. Deep resequencing of the full region would be required to identify such functional relevant variation.

Psoriasis is a chronic disorder in which T-cell-mediated inflammation causes thickening of the skin. Conversely, it has been also hypothesized that in psoriatic patients, the lack of control of the outer skin cells may lead to the greatly increased production of cells that characterizes psoriasis. This, in turn, may lead to an abnormality of the blood vessels and the inflammation characteristic of psoriasis. Another possibility is that epidermal skin cells fail to mature into the flat, thickened, "cornified" layer they are supposed to. As a result, the epidermis tries to produce more cells than usual leading to the thickened epidermis, which then leads to inflammation. ADAM proteins have a complex organization that includes a signal sequence and the following domains: pro, metalloprotease (including a zinc-binding sequence), disintegrin, cysteine-rich, epidermal growth factor, transmembrane, and cytoplasmic tail domains. The proteins have diverse functions which include adhesion, cell fusion, intracellular signaling and the shedding of the extracellular portion of proteins such as cytokines and growth factors, leading to the soluble forms of these proteins. Expression data suggest that ADAM33 is expressed in most human tissues, including skin [YOSHINAKA et ah, Gene, vo 1.282, p :227-236, 2002]. It is biologically plausible that ADAM33 is relevant to the development of psoriasis because it may be involved in the inflammatory response, or in cell-cell and cell-matrix interactions that are essential for the development and maintenance of a tissue; likewise, extracellular matrix proteolysis is an important contributor to skin remodeling, which when altered might ultimately lead to significant desquamation or, perhaps, absence of cell maturation.

To conclude with, this is the first report of an association between ADAM33 and psoriasis. Confirmation of our findings in different populations would represent an important development in understanding susceptibility to psoriasis, allergy, and closely related phenotypes. The importance of this observation should be evaluated by further delineating the biological role OΪADAM33 in psoriasis.

Claims

1. A method of testing a subject thought to have or be predisposed to having psoriasis which comprises the step of analyzing a biological sample from said subject for: j) detecting the presence of a SNP in the ADAM33 gene, which SNP is associated with psoriasis , and/or ii) analyzing the expression of the ADAM33 gene.

2. The method of claim 1, wherein the SNPs in the ADAM33 gene associated with psoriasis are selected in the group comprising rs512625 (nucleotide N at position 31 of SEQ ID NO:6, wherein allele A is associated to psoriasis), rs677044 (nucleotide N at position 31 of SEQ ID NO:7, wherein allele G is associated to psoriasis), rs597980 (nucleotide N at position 31 of SEQ ID NO:8, wherein allele T is associated to psoriasis), rs44707 (nucleotide N at position 31 of SEQ ID NO:9, wherein allele C is associated to psoriasis), rs628977 (nucleotide N at position 31 of SEQ ID NO:21, wherein allele T is associated to psoriasis), rs598418 (nucleotide N at position 27 of SEQ ID NO:22, wherein allele C is associated to psoriasis), rs2853209 (nucleotide N at position 27 of SEQ ID NO:23, wherein allele A is associated to psoriasis), rs2787095 (nucleotide N at position 27 of SEQ ID NO:24, wherein allele C is associated to psoriasis), rs2853213 (nucleotide N at position 27 of SEQ ID NO:25, wherein allele G is associated to psoriasis), and rs 1046919 (nucleotide N at position 27 of SEQ ID NO:26, wherein allele C is associated to psoriasis).

3. The method of any one of claim 1 or 2, wherein the SNPs in the ADAM33 gene associated with psoriasis are selected in the group comprising rs512625 (nucleotide N at position 31 of SEQ ID NO:6, wherein allele A is associated to psoriasis), rs677044 (nucleotide N at position 31 of SEQ ID NO:7, wherein allele G is associated to psoriasis), rs597980 (nucleotide N at position 31 of SEQ ID NO:8, wherein allele T is associated to psoriasis), rs44707 (nucleotide N at position 31 of SEQ ID NO:9, wherein allele C is associated to psoriasis).

4. The method of any one of claims 1 to 3, wherein the detection of single nucleotide polymorphisms is realized with a technique selected in the group comprising dynamic allele-specific hybridisation, ligation chain reaction, mini-sequencing, DNA"chips", allele-specific oligonucleotide hybridisation with single or dual-labelled probes merged with PCR or with molecular beacons, and others.

5. A method for treating and/or preventing psoriasis in a subject, comprising the administration of an effective amount of a compound which specifically inhibits the expression of ADAM33 gene to said subject.

6. A method for treating and/or preventing psoriasis in a subject, comprising the administration of an effective amount of a compound which specifically increases the expression of ADAM33 gene to said subject.

7. An in vitro method of selecting a compound, which can be useful for treating psoriasis, characterized in that said method comprises the steps of: e) obtaining a cell expressing ADAM33 gene; f) contacting said cell with at least one compound, g) comparing the expression of the ADAM33 gene in the cell between the steps a) and b), h) selecting the compound, which induces a lower level of expression of the ADAM33 gene in the cell contacted to that compound.

8. An in vitro method of selecting a compound, which can be useful for treating psoriasis, characterized in that said method comprises the steps of:

e) obtaining a cell expressing ADAM33 gene;

f) contacting said cell with at least one compound,

g) comparing the expression of the ADAM33 gene in the cell between the steps a) and b),

h) selecting the compound, which induces a higher level of expression of the ADAM33 gene in the cell contacted to that compound.