EP1981990A2 - Method for diagnosing of skin diseases caused by a mutation in the znf750 gene - Google Patents

Abstract

The invention provides a method for diagnosing a skin disease that is associated with the ZNF750 gene in an individual, comprising determining an aberration in the sequence or expression of the ZNF750 gene and/or determining the identity of single nucleotide polymorphism of the ZNF750 gene, in a biological sample of said individual.

Description

METHOD FOR DIAGNOSING OF SKIN DISEASES CAUSED BY A

MUTATION IN THE ZNF750 GENE

FIELD OF THE INVENTION

The present invention is in the field of molecular diagnostics and therapy of skin diseases, disorders or conditions that are associated with the ZNF750 gene.

BACKGROUND OF THE INVENTION Seborrheic dermatitis (SD) and Psoriasis, each affecting 2-3% of the population worldwide and up to 85% of AIDS patients, are distinct chronic papulosquamous dermatoses (Plewig et al, 1999; Gupta et al., 2004a; Braun-Falco et al., 1991). Psoriasis is characterized by red, scaly skin patches usually found on the scalp, elbows and knees, and may be associated with arthritis (Braun-Falco et al., 1991). Clinical manifestations of SD vary from common dandruffs to pityriasiform, eczematous or psoriasiform plaques with predilection for sebaceous follicle-rich areas on the scalp, face and trunk. Both diseases present with increased epidermal proliferation of keratinocytes and a sparse dermal perivascular infiltration of inflammatory cells (Faergemann et al., 2001). Follicular plugging by orthokeratosis and parakeratosis may be noted, as well as overgrowth of the saprophyte Pityrosporum (Gupta et a., 2004b). Occasionally, distinction between the two entities can be obscured by overlapping clinical and histopathological features. The molecular basis of both diseases is yet unclear (Bowcock et al., 2005) but they both involve enhanced proliferation of keratinocytes, skin infiltrates of cells of the immune system, and local over-growths of the saprophyte Pityrosporum ovale (known also as Malassezia furfur). The pathological over-lap between the two diseases is reflected also in a clinical overlap. Since both acne and Tinea versicolor are also characterized by involvement of keratinocytes and infestation with Pityrosporum, it is possible that the disease mechanism of acne and Tinea versicolor is, at least partially, common to that of psoriasis and/or SD. Currently there are only partial palliative treatments and no dependable molecular diagnostic tools available for these disease.

SUMMARY OF THE INVENTION

The present invention relates to method for diagnosing a disease, disorder or condition of the skin that is associated with the ZNF750 gene (SEQ ID NO: 1) in an individual, comprising determining the identity of a mutation in the ZNF750 gene sequence and/or detecting an aberration in the expression of the ZNF750 gene, in a biological sample of said individual. In a preferred embodiment, the method comprises determining the identity of a mutation in the ZNF750 gene sequence including, but not limited to, a duplication of the nucleotides CC (56_57dupCC), the mutation -347 A/C, the mutation -319C/T, or the mutation +46C/T.

The skin disease, disorder or condition associated with the ZNF750 gene is selected from psoriasiform seborrheic dermatitis, seborrheic dermatitis, atopic dermatitis, acne, tinea versicolor, or any of their variants or, preferably, psoriasis. Examples are given in Examples 1 and 2 below.

In one aspect, the present invention relates to a method for identifying a modulator of the ZNF750 gene expression comprising: (i) contacting keratinocytes with a candidate substance; (ii) measuring the transcription of the ZNF750 gene and/or translation of the ZNP750 transcript in the presence or the absence of said candidate substance, wherein a candidate substance capable of modulating the ZNF750 gene transcription and/or translation of the ZNP750 transcript is a modulator of the expression of the ZNF750 gene. A polynucleotide comprising a DNA sequence of the ZNF750 gene, a

ZNF750 cDNA (SEQ ID NO: 2), a gene homologous to the ZNF750 gene, or an antibody or compound specifically targeted at the ZNF750 protein (SEQ ID NO: 3) or its fragment(s) can be used as probes for specifically measuring the abundance of ZNF750 transcripts or ZNF750 protein. The invention further provides a method for identifying a modulator of the activity of a ZNF750 polypeptide comprising: (i) contacting a ZNF750 polypeptide comprising an amino acid sequence of SEQ ID NO: 3, a fragment or a variant thereof, with a candidate substance; (ii) detecting the complex formed between said polypeptide and said candidate substance; and (iii) measuring the activity of the ZNF750 polypeptide in the presence or the absence of said candidate substance capable of forming a complex with said ZNF750 polypeptide, wherein a candidate substance capable of forming a complex with said ZNF750 polypeptide and changing its activity is a modulator of the activity of said ZNF750 polypeptide. Further provided by the invention is a method for identifying a gene or protein involved in a skin disease, disorder or condition selected from psoriasiform seborrheic dermatitis, psoriasis, seborrheic dermatitis, their variants, acne, Tinea versicolor, atopic dermatitis and/or Pityrosporum infection, said gene or protein being regulated by the ZNF750 gene, comprising: (i) identifying differentially expressed or activated gene(s) or protein(s) in a cell culture exhibiting said disease, disorder or condition characteristics comprising keratinocytes obtained from an individual having said skin disease, disorder or condition and in a cell culture comprising normal keratinocytes; (ii) decreasing or increasing expression of the ZNF750 gene in said cell cultures by contacting the cell cultures with a modulator identified as described above; and (iii) determining the effect of decreasing or increasing expression of the ZNF750 gene on the expression or activity of said differentially expressed or activated gene(s) and/or protein(s) and on the disease characteristics exhibited by the cells of (ii), wherein a change in expression or activity of said differentially expressed or activated gene and/or protein and in the disease characteristics exhibited by said cells is indicative of said differentially expressed or activated gene and/or protein being regulated by the ZNF750 gene and being involved in a skin disease, disorder or condition. Preferentially, the activity of the ZNF750 gene product results in modulation of keratinocyte proliferation or cytokine production by keratinocytes. In one aspect, the invention relates to a method for identifying a gene or protein involved in modulation of Pityrosporum ovale infection, comprising: (i) identification of differentially expressed or activated gene(s) or protein(s) in a cell culture comprising keratinocytes from a sample of skin infected with Pityrosporum ovale exhibiting the characteristics of skin infection and in a cell culture comprising normal keratinocytes; (ii) decreasing or increasing expression or activity of the ZNF750 gene in said cell cultures by contacting the cell cultures with a modulator identified by methods described above herein; and (iii) determining the effect of decreasing or increasing expression of the ZNF750 gene on the expression or activity of said differentially expressed or activated gene(s) and/or protein(s) and on the skin infection characteristics exhibited by the cells of (ii), wherein a change in expression or activity of said differentially expressed or activated gene and/or protein and in the skin infection characteristics exhibited by said cells is indicative that said differentially expressed or activated gene and/or protein is regulated by the ZNF750 gene and is involved in modulation of the Pityrosporum ovale infection.

An additional aspect of the invention relates to a method for treatment of a disease, disorder or condition selected from psoriasiform seborrheic dermatitis, psoriasis, seborrheic dermatitis, their variants, acne, Tinea versicolor, atopic dermatitis and/or Pityrosporum infection, comprising administering to a patient in need a therapeutically effective amount of a molecule selected from: (i) a polynucleotide comprising a ZNF750 gene, a ZNF750 cDNA, a ZNF750 mRNA, a ZNF750 siRNA, a ZNF750 antisense DNA or RNA, a gene homologous to ZNF750, or a fragment thereof; (ii) a polypeptide comprising a ZNF750 protein, or a fragment thereof; (iii) an antibody specific to a ZNF750 polypeptide, and/or (iv) a modulator of ZNF750 gene transcription and/or translation of the ZNP750 transcript, and/or of ZNF750 protein function.

Yet an additional aspect of the invention relates to a method for testing the efficacy of a candidate compound for treating a disease, disorder or condition selected from psoriasiform seborrheic dermatitis, psoriasis, seborrheic dermatitis, their variants, acne, Tinea versicolor, atopic dermatitis and/or Pityrosporum infection, comprising contacting keratinocytes exhibiting said disease, disorder or condition characteristics in which the expression of the gene ZNF750 and/or the function of the ZNF750 protein has been partially or completely silenced with a candidate substance, whereby a candidate substance causing reduction in said disease characteristics exhibited by said cells is indicative of the efficacy of said candidate compound in treating said disease, disorder or condition.

A further aspect of the invention relates to a method for identifying a compound capable of modulating keratinocyte proliferation and/or cytokine production by keratinocytes, comprising contacting a candidate compound with keratinocytes, in which the expression of the gene ZNF750 and/or the function of the ZNF750 protein has been partially or completely silenced, and comparing keratinocyte proliferation and cytokine production by said keratinocytes with that of normal keratinocytes, wherein a candidate compound capable of modulating keratinocyte proliferation and/or cytokine production by said keratinocytes is a modulator of keratinocyte proliferation and/or cytokine production by keratinocytes.

In yet a further aspect, the invention provides a method for controlling cell proliferation in- vivo or in vitro comprising modulating the expression or function of the ZNF750 gene or protein. Also, a method for controlling cell proliferation in- vivo or in vitro comprising the use of ZNF750 gene, protein or derivative thereof, is contemplated.

In an additional aspect, the invention refers to the use of a molecule selected from: (i) a polynucleotide comprising a ZNF750 gene, a ZNF750 cDNA, a ZNF750 mRNA, a ZNF750 siRNA, a ZNF750 antisense DNA or RNA, a gene homologous to ZNF750, cDNA, mRNA, siRNA, antisense, homologous gene, or a fragment thereof; (ii) a polypeptide comprising a ZNF750 protein, or a fragment thereof; (iii) an antibody specific to a ZNF750 polypeptide; or (iv) a modulator of ZNF750 gene transcription and/or translation of the ZNP750 transcript, and/or of ZNF750 protein function, for the preparation of a pharmaceutical composition for the treatment of a disease, disorder or condition selected from psoriasiform seborrheic dermatitis, psoriasis, seborrheic dermatitis, their variants, acne, Tinea versicolor, atopic dermatitis and/or Pityrosporum infection.

In yet another aspect, the invention refers to a pharmaceutical composition for the treatment of a disease, disorder or condition selected from psoriasiform seborrheic dermatitis, psoriasis, seborrheic dermatitis, their variants, acne, Tinea versicolor, atopic dermatitis and/or Pityrosporum infection, comprising a molecule selected from: (i) a polynucleotide comprising a nucleotide sequence of the ZNF750 gene, a ZNF750 cDNA, a ZNF750 mRNA, a ZNF750 siRNA, a ZNF750 antisense DNA or RNA, a polynucleotide comprising a nucleotide sequence of a gene homologous to the ZNF750 gene , mRNA, siRNA, antisense, homologous gene, or a fragment thereof; (ii) a polypeptide comprising a ZNF750 protein, or a fragment thereof; (iii) an antibody specific to a ZNF750 polypeptide, and a pharmaceutically acceptable carrier; and/or (iv) a modulator of ZNF750 gene transcription and/or translation of the ZNP750 transcript, and/or of ZNF750 protein function.

BRIEF DESCRIPTION OF THE FIGURES

Figs, la-n show disease phenotype in affected individuals of a single extended family, that were demonstrated to carry the duplication of CC (56_57CC) in a single allele of the ZNF750 sequence. Fig. la-b show seborrhea-like facial erythema and scaling accentuated in supraorbital and perioral areas, with marked erythema and scaling of face. Fig. Ic shows hyperkeratotic hyperpigmented plaques over elbows. Fig. Id depicts hyperkeratotic erythematous plaques over knuckles and proximal interphalangeal joints. In Fig. Ie, pustules on the nape. In Fig. If, enlarged follicular ostium showing yeasts of Pityrosporum ovale (white arrows) (PAS stain x 400). In Figs, lg-h, papillomatosis, acanthosis, hyperkeratosis and mounds of parakeratosis (arrows), few ectatic blood vessels with mild perivascular mononuclear cell infiltrate (H&E xlOO; x400). In Fig. Ii, hair follicle showing keratin plugs and mounds of parakeratosis (arrows) on either side of the follicular ostium (H&E x200). In Figs, lj-k, perivascular lymphocyte infiltrate: most cells are CD4 (Fig. Ij), and only few are CD8 positive (Fig. Ik) (immunohistochemistry x400). Fig. 11 shows keratin 16 in affected skin (immunohistochemistry x200). In Figs, lm-n, Ki67 staining of normal (Fig. Im) vs. affected (Fig. In) skin (immunohistochemistry x400). The histological findings were observed in 4 of 4 affected individuals studied. All 4 individuals were of the same extended family and carried an identical ZNF750 mutation. Figs. 2a-b show further description of the disease phenotype in affected individuals in the family described in Fig. 1: In Fig. 2a, mild scaling and erythema on earlobe. In Fig. 2b, striate keratoderma over pressure sites on the palms and fingers. Few features of this dermatosis (nail dystrophy, dental carries and striate keratoderma over pressure sites) are not characteristic of psoriasis or SD. Figs. 3a-g show family tree and linkage analysis in a multigenerational family presenting with autosomal dominant inheritance of the disease phenotype that is given in Fig. 1 and Fig. 2, and analysis of gene expression of ZNF750. Fig. 3a depicts fine mapping: Genomic location of the ZNF750 CC duplication (wild type vs. mutant alleles). Figs. 3b-c depicts ZNF750 expression in normal human tissues and cells: RT-PCR products of (Fig. 3b) ZNF750 ex 1-2 (210bp) (SEQ ID NOs: 4-5) and (Fig. 3c) reference gene GAPDH (200bp) (SEQ ID NOs: 6-7). RT- PCR amplification of ZNF750 from activated and non-activated T cells was minimal (visible amplification product achieved only after two sequential rounds of RT-PCR). Real-time PCR showed near zero copy number of ZNF750 transcripts in both activated and non-activated CD4 cells (not shown). Fig. 3d shows RT-PCR from mRNA of keratinocytes of affected (Aff.l, Aff.2) and non-affected (Cont.l, Cont.2) individuals, using primers specific to the normal allele and to the mutant allele (SEQ ID NOs: 8-11). Amplicons of expected sizes, sequences verified. (Figs. 3e-f). Real-time qRT-PCR amplification of ZNF750 mRNA, using primer sets unique (Fig. 3e) or common (Fig. 3f) to the mutant and the native ZNF750 alleles. Results normalized to an internal housekeeping control gene (GAPDH), shown not to vary between samples. Fig. 3e shows that keratinocytes of affected individuals harbor nearly equal copy numbers of ZNF750 native and mutant transcripts: copy number of mutant transcripts divided by copy number of native transcripts (mean ± s.e., n=3). Fig. 3f shows that keratinocytes of affected individuals harbor nearly double the copy number of ZNF750 transcripts as compared to non-affected individuals: copy number of ZNF750 transcripts in keratinocytes of 3 affected individuals divided by the copy number in keratinocytes of 3 controls (mean ± s.e). Fig. 3g shows peripheral blood CD4 T lymphocyte proliferation assay in affected vs. non-affected individuals (mean ± s.e for triplicates of each sample, n=6).

Figs. 4a-e show mutation analysis of ZNF750 exon 2 in affected individuals whose disease phenotype is given in Fig. 1 and Fig. 2. DNA sequence of ZNF750 in (Fig. 4a) Control (unaffected) individual, (Fig. 4b) Affected individual and (Fig. 4c) mutant allele from affected individual subcloned into pGEM-T vector. In the affected individuals there is a duplication of CC (56_57dupCC) that was verified by subcloning of the mutant allele. Fig. 4d shows single peaked pattern observed on denaturing high-performance liquid chromatography analysis of control (unaffected) sample (i) as compared to double peaked pattern observed in sample of an affected individual (ii). Fig. 4e shows alignment of the ZNF750 homologues in various organisms. C2H2 zinc finger domain is boxed.

Figs. 5a-c depict sequence analysis of ZNF750 promoter region and 5' untranslated region in individuals with "classical" psoriasis: DNA sequence in affected individuals with heterozygous single nucleotide changes -347A/C (Fig. 5a), -319C/T (Fig. 5b), +46C/T (Fig. 5c). Figs. 6a-d show DHPLC analysis of ZNF750 promoter region and 5' untranslated region in individuals with "classical" psoriasis: DHPLC analysis results in three unrelated affected individuals with single nucleotide change +46C/T (Fig. 5a-c) and in an affected individual with the -319C/T single nucleotide change (Fig.5b). Each figure demonstrates the DHPLC result for the affected individual (upper chromatograph with two peaks) in comparison with the normal DHPLC chromatograph of the equivalent PCR amplicons in control healthy individuals (lower chromatograph in each figure, with single peak).

Fig. 7 shows a luciferase-reporter assay testing the effect of the +46.C/T single nucleotide change (found in "classical" psoriasis patients) on ZNF750 promoter activity. Four constructs, containing segments -750 to +78 of the ZNF750 promoter upstream of the luciferase reporter gene in the promoter-less pGL3 basic vector, were transiently transfected into HEK293 cell line along with Renilla plasmid that served as an internal control for transfection efficiency. pGL3 p.-750 wt Clone A and Clone B, transfection with two different luciferase reporter plasmids identical in sequence, carrying the wild type ZNF750 promoter segment; ρGL3 p.-750 mut+46 (SEQ ID NO: 12), transfection with a luciferase reporter plasmid carrying the mutant +46C/T segment; pGL3 Basic only, transfection with the luciferase reporter plasmid with no promoter (negative control); TK mini promoter, transfection with the luciferase reporter plasmid carrying a control promoter not related to ZNF750 (positive control). Results are given as mean of 3 repeat experiments +SD (standard deviation).

Fig. 8 shows 5' RACE analysis of ZNF750 transcripts in normal human keratinocytes. Four PCR amplicons were generated (A₃B, C,D). Sequence analysis demonstrated that only two of the PCR amplicons (A, C) contain ZNF750 sequences . "A" represents the ZNF750 transcript described in example 1 (SEQ ID NO: 14). ^UC" represents a longer transcript (SEQ ID NO: 15) encoding a putative 46 amino acid polypeptide.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the following aspects: (i) any use of the

ZNF750 gene, mRNA, small interfering RNA (siRNA), antisense, homologous gene, mutant, fragment thereof or the encoded protein product or their segments, homologues or their derivatives, or antibodies to any segments or derivatives of these molecules, in the diagnosis of psoriasis, atopic dermatitis, seborrheic dermatitis or any of their variants; (ii) any therapy manipulating interactions or function of the ZNF750 gene, its mRNA, or encoded protein, or their segments, homologues or derivatives, with other molecules such as DNA, mRNA, or proteins), for the treatment of psoriasis, seborrheic dermatitis or any of their variants, acne, Tinea versicolor, atopic dermatitis and/or Pityrosporum infection; and (iii) any gene or protein regulated by the ZNF750 protein and/or any modulators of the activity of the ZNF750 protein or gene to treat or diagnose a skin disease disorder or condition in which the activity of the ZNF750 gene/protein is involved such as psoriasis, atopic dermatitis, seborrheic dermatitis, their variants.

It has been found in accordance with the present invention that the human ZNF750 (FLJ13841) gene (SEQ ID NO. 1), previously named by us "Psorael", is directly associated with psoriasiform seborrheic dermatitis (SD). Disclosed in the appendix below is the GeneCard for the FLJl 3841 gene (www.genecards.org). The FLJ 13841 protein contains a putative C2H2-like zinc finger domain and a nuclear localization signal. As disclosed above, atopic keratitis and seborrheic dermatitis share many clinical manifestations. Also, atopic dermatitis has similarities to seborrheic dermatitis in its immune mechanism, and has been associated with the genomic locus 17q25 (Strausberg et al., 200; Ota et al., 2004) and the PSORS2 locus (Morar et al., 2006; Bowcock et al., 2004; Speckman et al., 2003; Cookson et al., 2001), in which the FLJ 13841 gene resides. As we have identified ZNF750 as the disease- associated gene within the PSORS2 locus, our results indicate an association of ZNF750 with atopic dermatitis. Thus, there is a possibility that FLJ13841 might play a role in this phenotype too.

In one aspect the invention relates to a method for diagnosing a disease, disorder or condition of the skin that is associated with the ZNF750 gene (SEQ ID NO. 1) in an individual, comprising determining the identity of a mutation in the ZNF750 gene sequence and/or detecting an aberration in the expression of the ZNF750 gene, in a biological sample of said individual.

In a preferred embodiment, the skin disease, disorder or condition associated with the ZNF750 gene is selected from psoriasiform seborrheic dermatitis, seborrheic dermatitis, atopic dermatitis, acne, tinea versicolor, or any of their variants or, most preferably, psoriasis.

The mutation in the ZNF750 gene sequence includes, but is not limited to, single or multiple nucleotide exchange, deletion of one or more nucleotides or insertion of one or more nucleotides in a nucleotide sequence In a preferred embodiment, the aberration in the sequence of the ZNF750 gene is a duplication of the nucleotides CC (56_57duρCC), the mutation -347 ^'AJC, the mutation -319C/T or the mutation +46C/T.

The genomic DNA used for the diagnosis may be obtained from body cells, such as those present in peripheral blood, urine, saliva, tissue biopsy, OR surgical specimen.

A skilled artisan recognizes that there are a variety of methods to detect a mutation in a nucleic acid sequence. For example, methods regarding allele-specific probes for analyzing particular nucleotide sequences are described by e.g., Saiki et al, 1986; US 836,378; US 943,006; US 197,000; and US 347,495. Allele-specific probes are typically used in pairs. One probe (primer) shows perfect complementarity to a wild type allele and the other probe is complementary to a variant allele. The primers are used in conjunction with a second primer, which hybridizes at a distal site. PCR amplification proceeds from the two primers leading to a detectable product signifying the particular allelic form is present.

Alternatively, in order to effectively separate mixtures of double stranded polynucleotides in general, and DNA in particular, based on base pair length, a new technology called Ion-pair reverse-phase high pressure liquid chromatography (IP- RP-HPLC), also referred to as Matched Ion Polynucleotide Chromatography (MIPC), was introduced recently. When IP-RP-HPLC analyses were carried out at a partially denaturing temperature, i.e., a temperature sufficient to denature a heteroduplex at the site of base pair mismatch, homoduplexes could be separated from heteroduplexes having the same base pair length (Underhill, et al., 1996). Thus, the use of denaturing high performance liquid chromatography (DHPLC) was applied to mutation detection (Underhill, et al., 1997; Liu, et al., 1998). These chromatographic methods are generally used as a first line of screening to detect whether or not a mutation exists in a test DNA fragment. DNA fragments with different DHPLC chromatograms are then sequenced to reveal the nature of the DNA alteration. DHPLC had been shown to meet these criteria for a growing number of applications in disease-related genes (Bercovich and Beaudet, 2003). Scanning for DNA mutations and variants using dHPLC involves subjecting PCR products to chromatography using an ion-pair reversed-phase cartridge. PCR products are denatured and allowed to reanneal. Under conditions of partial denaturation with a linear acetonitrile gradient, heteroduplexes from PCR samples having an internal sequence variation display a reduced column retention time relative to their homoduplex counterparts. The elution profile for heterozygous samples is typically quite distinct from that of either homozygous sequence, making the identification of heterozygous mutations relatively straightforward.

Particular nucleic acid sites can also be identified by hybridization to oligonucleotide arrays. An example is described in WO 95/11995.

A polynucleotide comprising a nucleotide sequence of the ZNF750 gene, a ZNF750 cDNA, or a DNA sequence homologous to the ZNF750 gene, or a fragment thereof, may be used as diagnostic reagents to detect the presence or absence of the ZNF750 gene DNA or RNA sequences to which they specifically bind, such as for determining the level of expression of the gene or for determining whether the gene contains a genetic aberration.

The term homologous is used herein to describe a heterologous DNA sequence that can be used as a probe to detect the native or mutated ZNF750 gene or RNA transcript under appropriate hybridization conditions. That is, the percent identity of the homologous gene to the native gene may vary from almost identical to substantially different, as long as, under appropriate stringency, the homologous gene is capable of specifically hybridize to and detect the ZNF750 gene or RNA transcript.

A fragment of a nucleotide sequence is defined herein as a section of a whole nucleotide sequence that retains the essential function(s) of the whole nucleotide sequence. A fragment of a polypeptide sequence is defined herein as a section of a whole polypeptide sequence that retains all or some of the essential function(s) of the whole polypeptide sequence. The term mutation is used herein interchangeably with the term genetic aberration and includes, but is not limited to, single or multiple nucleotide exchange, deletion of one or more nucleotides or insertion of one or more nucleotides in a nucleotide sequence. The term ZNF750 gene refers both to the DNA sequence of the wild type gene found in healthy individuals and to an altered DNA sequence of the ZNF750 gene, including, but not limited to single or multiple nucleotide exchange, deletion of one or more nucleotides or insertion of one or more nucleotides in a nucleotide sequence. The siRNA comprise a sense RNA strand and a complementary antisense

RNA strand annealed together by standard Watson-Crick base-pairing interactions (hereinafter "base-paired"). As is described in more detail below, the sense strand comprises a nucleic acid sequence which is identical to a target sequence contained within the target mRNA. The sense and anti-sense strands of the above mentioned siRNA can comprise two complementary, single-stranded RNA molecules or can comprise a single molecule in which two complementary portions are base-paired and are covalently linked by a single-stranded "hairpin" area. Without wishing to be bound by any theory, it is believed that the hairpin area of the latter type of siRNA molecule is cleaved intra-cellularly by the "Dicer" protein (or its equivalent) to form an siRNA of two individual base-paired RNA molecules.

As used herein, siRNA refers to "isolated siRNA" meaning altered or removed from the natural state through human intervention. For example, an siRNA naturally present in a living animal is not "isolated," but a synthetic siRNA, or an siRNA partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated siRNA can exist in substantially purified form, or can exist in a non-native environment such as, for example, a cell into which the siRNA has been delivered. As used herein, the term gene expression refers to both the process of transcription of the gene to produce an mRNA transcript and to the process of translation of the transcript on the ribosomes to produce a polypeptide product.

In one aspect, the present invention relates to a method for identifying a modulator of the ZNF750 gene expression comprising: (i) contacting keratinocytes with a candidate substance; (ii) measuring the transcription of the ZNF750 gene and/or translation of the ZNP750 transcript in the presence or the absence of said candidate substance, wherein a candidate substance capable of modulating the ZNF750 gene transcription and/or translation of the ZNP750 transcript is a modulator of the expression of the ZNF750 gene. A polynucleotide comprising a ZNF750 cDNA, or a DNA sequence homologous to the ZNF750 gene, or a fragment thereof, can be used as probes for specifically measuring the abundance of ZNF750 transcripts. An antibody targeted at ZNF750 protein or its fragments can be used to measure the abundance of ZNF750 protein. An additional aspect relates to a method for identifying a modulator of the activity of a ZNF750 polypeptide comprising: (i) contacting a ZNF750 polypeptide comprising an amino acid sequence of SEQ ID NO: 2, a fragment or a variant thereof, with a candidate substance; (ii) detecting the complex formed between said polypeptide and said candidate substance; and (iii) measuring the activity of the ZNF750 polypeptide in the presence or the absence of said candidate substance capable of forming a complex with said ZNF750 polypeptide, wherein a candidate substance capable of forming a complex with said ZNF750 polypeptide and changing its activity is a modulator of the activity of said ZNF750 polypeptide.

In one embodiment, a modulator of the ZNF750 gene expression is activating ZNF750 gene expression or increasing the ZNF750 polypeptide activity and thus fully or partially reverses or attenuates clinical manifestations observed in patients with mutated ZNF750 gene or down-regulated ZNF750 gene product. In contrast, a modulator of the ZNF750 gene expression is down-regulating ZNF750 gene expression or decreasing the ZNF750 polypeptide activity and thus is useful for identifying genes regulated by the ZNF750 gene product. Examples of candidate substances include, but are not limited to, a synthetic peptide from a library of peptides, an antibody, and a natural polypeptide from a cell extract or biological fluid. In one embodiment of the invention, the activity of the ZNF750 polypeptide is modulating keratinocyte proliferation. In another embodiment of the invention, the activity of the ZNF750 polypeptide is modulating cytokine production by keratinocytes.

One example of a method for detecting the complex formed between said polypeptide and said candidate substance is immunoprecipitation, in which the candidate compound is labeled with a fluorescent or radioactive probe and contacted with the polypeptide. The compound-polypeptide mixture is then contacted with an antibody specific for the polypeptide that is attached to a magnetic or polymeric bead, and the immunocomplex is precipitated by magnetic or centrifugal force. The presence of label in the precipitate indicates that a complex has formed between the labeled candidate compound and the polypeptide. One object of the invention is to provide a method for identifying a gene or protein involved in a skin disease, disorder or condition selected from psoriasiform seborrheic dermatitis, psoriasis, seborrheic dermatitis, their variants, acne, Tinea versicolor, atopic dermatitis and/or Pityrosporum infection, said gene or protein being regulated by the ZNF750 gene, comprising: (i) identifying differentially expressed or activated gene(s) or protein(s) in a cell culture exhibiting said disease, disorder or condition characteristics comprising keratinocytes obtained from an individual having said skin disease, disorder or condition and in a cell culture comprising normal keratinocytes; (ii) decreasing or increasing expression of the ZNF750 gene in said cell cultures by contacting the cell cultures with a modulator of claim 4; and (iii) determining the effect of decreasing or increasing expression of the ZNF750 gene on the expression or activity of said differentially expressed or activated gene(s) and/or protein(s) and on the disease characteristics exhibited by the cells of (ii), wherein a change in expression or activity of said differentially expressed or activated gene and/or protein and in the disease characteristics exhibited by said cells is indicative of said differentially expressed or activated gene and/or protein being regulated by the ZNF750 gene and being involved in a skin disease, disorder or condition.

In one embodiment the activity of the ZNF750 gene product results in modulation of keratinocyte proliferation or cytokine production by keratinocytes; and thus, the modulation of keratinocyte proliferation or cytokine production by keratinocytes is measured in the absence or presence of the candidate substance, and a substance that causes an increase or decrease in these parameters and has been shown to form a complex with the ZNF75O polypeptide is a modulator of the ZNF750 polypeptide activity. The effectiveness of tested compounds in cell cultures delineated above can be assayed by measurements such as, but not limited to, proliferation rate assays (measured by cell counts, bromodeoxiuridine incorporation measurements, etc.) and/or assays of cytokine production (measured by methods such as quantitative realtime PCR, gene expression microarrays or ELISA of cell extracts or ELISA of cell culture medium).

Pityrosporum ovale is a saprophyte found abundantly in lesions of psoriasis, seborrheic dermatitis, as well as acne and Tinea versicolor. Antibiotic treatment of Pityrosporum ovale is known to improve the clinical phenotype of all these skin disorders. The mechanism that predisposes people with these skin disorders to have infestation with Pityrosporum ovale is unknown.

Pityrosporum ovale was identified in excessive quantities in disease lesions of affected individuals in the family that we studied herein (example 1 below), which have the mutation in the ZNF750 gene. The ZNF750 gene may be involved in the mechanism that predisposes people with these skin disorders to have Pityrosporum ovale infection. Therefore, the ZNF750 gene/protein may be instrumental for the identification of molecular or immune mechanisms predisposing to or prolonging/sustaining Pityrosporum ovale infection. Thus, the ZNF750 gene/protein may be helpful for identifying novel ways of combating/preventing Pityrosporum ovale infection, and therefore for the treatment or prevention of diseases associated with Pityrosporum ovale infection such as psoriasis, seborrheic dermatitis, acne and Tinea versicolor. In addition, the ZNF750 gene or protein may be used to identify novel ways of diagnosing diseases that are caused by Pityrosporum ovale infection.

Thus, in one aspect, the invention relates to a method for identifying a gene or protein involved in modulation of Pityrosporum ovale infection, comprising: (i) identification of differentially expressed or activated gene(s) or protein(s) in a cell culture comprising keratinocytes from a sample of skin infected with Pityrosporum ovale exhibiting the characteristics of skin infection and in a cell culture comprising normal keratinocytes; (ii) decreasing or increasing expression or activity of the ZNF750 gene in said cell cultures by contacting the cell cultures with a modulator of claim 4; AND (iii) determining the effect of decreasing or increasing expression of the ZNF750 gene on the expression or activity of said differentially expressed or activated gene(s) and/or protein(s) and on the skin infection characteristics exhibited by the cells of (ii), wherein a change in expression or activity of said differentially expressed or activated gene and/or protein and in the skin infection characteristics exhibited by said cells is indicative that said differentially expressed or activated gene and/or protein is regulated by the ZNF750 gene and is involved in modulation of the Pityrosporum ovale infection.

An additional aspect of the invention refers to a method for treatment of a disease, disorder or condition selected from psoriasiform seborrheic dermatitis, psoriasis, seborrheic dermatitis, their variants, acne, Tinea versicolor, atopic dermatitis and/or Pityrosporum infection, comprising administering to a patient in need a therapeutically effective amount of a molecule selected from: (i) a polynucleotide comprising a nucleotide sequence of the ZNF750 gene, a ZNF750 cDNA, a ZNF750 mRNA, a ZNF750 siRNA, a ZNF750 antisense DNA or RNA, a polynucleotide comprising a nucleotide sequence of a gene homologous to the ZNF750 gene, or a fragment thereof; (ii) a polypeptide comprising a ZNF750 protein, or a fragment thereof; (iii) an antibody specific to a ZNF750 polypeptide, and/or (iv) a modulator of ZNF750 gene transcription and/or translation of the ZNFP750 transcript, and/or of ZNF750 protein function. A polynucleotide comprising a ZNF750 cDNA, or a DNA sequence homologous to ZNF750, or a fragment thereof, may be used to treat an individual having a skin disease, disorder or condition as mentioned above, by incorporating said polynucleotide in an expression vector, viral vector or any other expression construct for expression of genes in eukaryotic cells known in the arts, administering this expression construct to a person in need so that the foreign ZNF750 polynucleotide is expressed in keratinocytes at a level sufficient to overcome the phenotype caused by the aberration in the in native ZNF750 gene of the person in need. A polynucleotide comprising a ZNF750 siRNA or a ZNF750 antisense polynucleotide may be used to treat an individual having a skin disease, disorder or condition as mentioned above, and who has one copy of the mutated ZNF750 gene, by expressing in keratinocytes of the individual in need mutant ZNF750 siRNA or ZNF750 antisense complementary to the ZNF750 sequence with an aberration. The mutant ZNF750 siRNA causes the degradation of the endogenous aberrant mRNA. The mutant ZNF750 antisense inhibits the translation of the endogenous aberrant mRNA. In both cases the transcript with the wild type sequence is translated into a normally functional polypeptide.

Yet an additional aspect of the invention refers to a method for testing the efficacy of a candidate compound for treating a disease, disorder or condition selected from psoriasiform seborrheic dermatitis, psoriasis, seborrheic dermatitis, their variants, acne, Tinea versicolor, atopic dermatitis and/or Pityrosporum infection, comprising contacting keratinocytes exhibiting said disease, disorder or condition characteristics in which the expression of the gene ZNF750 has been partially or completely silenced with a candidate substance, whereby a candidate substance causing reduction in said disease characteristics exhibited by said cells is indicative of the efficacy of said candidate compound in treating said disease, disorder or condition. The partially or complete silencing of the ZNF750 gene may be accomplished by exposing the keratinocytes to ZNF750 siRNA As mentioned, it has been found in accordance with the present invention that keratinocyte proliferation is enhanced in patients suffering of seborrhea-like dermatitis caused by the mutation in ZNF750. These results indicate that ZNF750 gene/protein is involved in modulation of keratinocytes proliferation Thus, yet another aspect refers to a method for identifying a compound capable of modulating keratinocyte proliferation and/or cytokine production by keratinocytes, comprising contacting a candidate compound with keratinocytes, in which the expression of the gene ZNF750 has been partially or completely silenced, and comparing keratinocyte proliferation and cytokine production by said keratinocytes with that of normal keratinocytes, wherein a candidate compound capable of modulating keratinocyte proliferation and/or cytokine production by said keratinocytes is a modulator of keratinocyte proliferation and/or cytokine production by keratinocytes.

A compound that decreases or increases keratinocyte proliferation and/or cytokine production is a modulator of keratinocyte proliferation and/or cytokine production. In one embodiment, a compound capable of modulating keratinocyte proliferation is reducing keratinocyte proliferation and thus ameliorates the increased epidermal proliferation of keratinocytes which is one of the hallmarks of Seborrheic dermatitis (SD) and Psoriasis . Methods for partially or completely silencing specific genes in mammals are well known in the art. For example, a gene may be deleted from the genome of the mammal as explained below.

There are two basic types of animals with genetically manipulated genomes. A traditional transgenic mammal has a modified gene introduced into its genome and the modified gene can be of exogenous or endogenous origin. This gene can encode for an siRNA that reduces the expression of a specific gene.

A "knock-out" mammal is a special type of transgenic mammal, characterized by suppression of the expression of an endogenous gene through genetic manipulation. The disruption of specific endogenous genes can be accomplished by deleting some portion of the gene or replacing it with other sequences to generate a null allele. Cross-breeding mammals having the null allele generates a homozygous mammals lacking an active copy of the gene. Such "knock-out" mammals can be generated so that the expression of the endogenous gene is suppressed in the entire animal throughout embryogenesis and its post-natal life, or in a conditional form - exhibiting suppression of the endogenous gene only in specific tissues or at a specific time.

A number of such mammals have been developed, and are extremely helpful in medical development. For example, U.S. Pat. No. 5,616,491 describes knock-out mice having suppression of CD28 and CD45. In yet a further aspect, a method for controlling cell proliferation in- vivo or in vitro comprises modulating the expression or function of the ZNF750 gene, protein or derivative thereof. Also, a method for controlling cell proliferation in- vivo or in vitro comprising the use of ZNF750 gene, protein or derivative thereof, is contemplated. In an additional aspect the invention refers to the use of a molecule selected from: (i) a polynucleotide comprising a nucleotide sequence of the ZNF750 gene, a ZNF750 cDNA, a ZNF750 mRNA, a ZNF750 siRNA, a ZNF750 antisense DNA or RNA, a polynucleotide comprising a nucleotide sequence of a gene homologous to the ZNF750 gene, mRNA, siRNA, antisense, homologous gene, or a fragment thereof; (ii) a polypeptide comprising a ZNF750 protein, or a fragment thereof; (iii) an antibody specific to a ZNF750 polypeptide, for the preparation of a pharmaceutical composition for the treatment of a disease, disorder or condition selected from psoriasiform seborrheic dermatitis, psoriasis, seborrheic dermatitis, their variants, acne, Tinea versicolor, atopic dermatitis and/or Pityrosporum infection; and/or a modulator of ZNF750 gene transcription and/or translation of the ZNP750 transcript, and/or of ZNF750 protein function.

In yet another aspect, the invention refers to a pharmaceutical composition for the treatment of a disease, disorder or condition selected from psoriasiform seborrheic dermatitis, psoriasis, seborrheic dermatitis, their variants, acne, Tinea versicolor, atopic dermatitis and/or Pityrosporum infection, comprising a molecule selected from: (i) a polynucleotide comprising a nucleotide sequence of the ZNF750 gene, a ZNF750 cDNA, a ZNF750 mRNA, a ZNF750 siRNA, a ZNF750 antisense DNA or RNA, a polynucleotide comprising a nucleotide sequence of a gene homologous to the ZNF750 gene, mRNA, siRNA, antisense, homologous gene, or a fragment thereof; (ii) a polypeptide comprising a ZNF750 protein, or a fragment thereof; (iii) an antibody specific to a ZNF750 polypeptide, and a pharmaceutically acceptable carrier; and/or (iv) a modulator of ZNF750 gene transcription and/or translation of the ZNP750 transcript, and/or of ZNF750 protein function.

Identification of the ZNF750 gene as the primary gene defective in psoriasiform SD is the basis for molecular analysis of the pathways through which SD and psoriasis or other skin diseases involving the ZNF750 gene evolve, and possibly for novel treatment modalities of these common disorders.

EXAMPLES

Methods

Clinical details. This study was approved by the Institutional Review Board of Soroka Medical Center and by the Israeli National Genetics Helsinki Committee. Written informed consent was obtained from all adult subjects and from parents on behalf of their children. Linkage and haplotype analysis. Genomic DNA was extracted from whole blood using standard procedures. Genome-wide linkage analysis was undertaken on DNA samples, using the ABI PRISM Linkage Mapping Set MDlO (Applied Biosystems). Four hundred fluorescent-labeled microsatellite markers, spaced at ~10cM intervals, were amplified from genomic DNA by PCR, per manufacturers' instructions. Products were separated by electrophoresis on an ABI PRISM 377 DNA Sequencer (Applied Biosystems), and analyzed using GeneScan software. Fine-mapping was carried out using polymorphic markers as follows: PCR products were separated on a 6% polyacrylamide gel, and visualized by silver-staining. Haplotypes were manually constructed and analyzed. The NCBI Build number for the physical map positions is 35.1. Statistical analysis: Statistical analysis was done using an autosomal dominant disease model assuming complete penetrance in both sexes. As the allele frequencies of the markers are not known in this particular study, we calculated the LOD (log of odds) score by assuming equal frequencies. Extended multipoint and two-point linkage analysis on the entire kindred was performed using SUPERLESfK (Fishelson and Geiger, 2002). The maximum multipoint lod score (8.79 at θ=0 for SNP rs3744165) was obtained when jointly using marker data at markers D17S784, rs869190, D17S928, rs4986110, and rs3744165. Other marker combinations including rs3744165 also yielded LOD scores higher than 5. Mutation analysis. The coding sequences of the 12 genes and 6 ESTs within the defined locus were amplified (SEQ ID NOs: 16-97) from cDNA generated from RNA that was extracted from EBV-transformed lymphoblastoid cells prepared from an affected individual. PCR primers were designed using the NCBI database and PRIMER 3 software. PCR products were subject to agarose-gel electrophoresis and gel extraction (QIAGEN), followed by sequencing with either the forward or reverse primer on an ABI PRISM 377 DNA Sequencer (Applied Biosystems). Results were analyzed using Chromas software, and the DNA sequences obtained were compared to published sequences. The PCR primers used for sequencing are shown in Table 1. Denaturing high-performance liquid chromatography (dHPLC) analysis of

ZNF750 exon 2 (SEQ ID NOs: 98-99) was carried out on genomic DNA samples using Transgenomic WAVE system, per manufacturer's protocol. PCR primers: in Table 1.

PCR products were eluted from the column using an acetonitrile gradient in a 0.1 M triethylamine acetate buffer (pH 7), at a constant flow rate of 0.9 ml min^"1. The temperature at which heteroduplex detection occurred was determined based on the melting profile of the specific DNA fragments, using WaveMaker software (Transgenomic). Fragments were analyzed with a total run time of 7.8 min per sample. The linear acetonitrile gradient was adjusted so that the peaks were eluted between 4.5 and 5.5 min Histochemical stainings and immunohistochemistry. Periodic acid-Schiff (PAS) stain, Hematoxillin Eosin and immunohistochemistry were done using standard techniques (Faergemann et al., 2001). Antibodies were purchased from Sigma. Analysis of ZNF750 transcripts in various tissues. A 210 bp fragment of

ZNF750 cDNA was amplified from human multiple tissue cDNA panels (Clontech), using specific primers (SEQ ID NO: 4-5) labeled "Exons 1-2" in Table 1. PCR products were subject to 2% agarose-gel electrophoresis.

T cell assays'. CD4 T cells were purified from peripheral blood using magnetic beads positive isolation kit (Dynal Biotech). The cells were cultured at a density of 1-2x10⁶ cells/ml in Isacove^Λs Modified Dulbecco's medium (Sigma) enriched with 10% fetal calf serum (FCS). Cell activation was performed using Phorbol Myristate Acetate (PMA, 100ng/ml) and Ionomycin (100ng/ml). T cell proliferation was tested with labeled thymidine as previously described (Cohen- Sfady et al., 2005). Results are shown as stimulation index for each of 3 affected and 3 non-affected individuals. Stimulation index is given as counts per minute (cpm) following induction divided by cpm without induction. Average background (non-activated) counts were 38, 226, 120, 88, 71, 369 for samples of affected individuals 1, 2, 3 and non-affected individuals 1, 2, 3, respectively. Real time quantitative PCR analysis. Total RNA was prepared using

RNAsol (Omega, Bio-Tek). Following DNase I (RNase free; Qiagene) digestion to remove genomic DNA, we used the ABgene cDNA synthesis kit containing both oligo(dT) and random hexamer primers to synthesize cDNA from 0.5μg of total RNA. The following reaction mixture was used for all PCR samples: 2 X sybrGreen supermix (ABgene, Epsom, UK), 100 to 200 nM of each primer, and 2.5 μl of cDNA in a 15μl total volume. Reactions were amplified and analyzed in triplicate using a Rotor-Gene RG-3000 real-time PCR detection system (Corbett Research, Mortlake, Australia).

The sense (F) and antisense (R) primers used for PCR amplification of ZNF750 exons 1-2 (SEQ ID NOs: 4-5) and 2-3 (SEQ ID NOs: 100-101) (Fig. 3f) were designed so that they were common to the mutant and the native transcript. The PCR primers used for Figs. 3d-e (termed Mutant and Native in the primer list) were designed to be specific for the mutant and the native ZNF750 transcripts (exon 2) (SEQ ID NOs: 8-11). The real-time RT-PCR mRNA levels were normalized to human GAPDH mRNA levels (SEQ ID NOs: 6-7).

Luciferase assay: DNA plasmids were generated so that segments from -750 to +78 of the ZNF750 gene sequence from a wildtype individual and from a psoriasis patient with the +46C/T mutation were cloned into the pGL3 luciferase reporter plasmid. ρGL3 luciferase reporter plasmid with no promoter servedas a negative control. pGL3 luciferase reporter plasmid carrying the TK mini promoter served as a positive control. These 4 constructs were transiently transfected into HEK293 cell line along with Renilla plasmid that served as an internal control for transfection efficiency. Luciferase activity was tested using standard methodology. Technical details of the experiment are as previously described (Glick et al., 2000). 5' SACE: To identify any further ZNF? ⁷50 transcripts besides the original one we described, a 5' RACE (Rapid Amplification cDNA Ends) was performed using BD™ SMART RACE cDNA Amplification Kit (BD Biosciences, Palo Alto, CA). The 5' RACE reaction was done on normal human keratinocyte mRNA. The ZNF750 specific primers for 5' RACE amplifications were primers X-GSP-R. To assure ZNF750 sequence specificity and for amplification of the RACE sequences, PCR primers 5'-RACE-GSP-R were applied to the initial RACE products in a further round of PCR. Four different RACE products (Fig. 8, A-D) were cloned into the pGEM®-T Easy vector (Promega, Madison, WI) and the sequences were determined. Nucleotide sequences were subjected to BLASTN and BLASTX searches of the GenBank rat resources database

(http://www.ncbi.nlm.nih.gov/genome/guide/rat/; Build 3.1) using default parameters and filters. Alignment and nucleotide sequence analysis of 50 clones revealed the original transcript, as well as a second less abundant transcript with an extension of ~500bp at the 5' end of exon 1 of the original transcript, encoding a putative ~46aa protein. All single nucleotide changes / mutations described above are 3' to the stop codon of the second, less abundant transcript, and are therefore less likely to have an effect on this transcript.

Table 1. PCR primers.

to

EXAMPLE 1.

A Jewish family of Moroccan descent presented with an apparently autosomal dominant form of seborrhea-like dermatosis with psoriasiform elements, affecting 44 individuals in 5 generations (Fig. 2). All affected family members presented by 10 years of age with a similar phenotype (Fig. 1; Fig. 2; Table 2): a chronic fine diffuse scaly erythematous rash on the face, particularly on the chin, nasolabial folds, eyebrows, around earlobes and over the scalp. The rash exacerbated in the winter, with emotional stress, and after strenuous physical activity. Hyperkeratosis of skin over the elbows, knees, palms, soles and metacarpophalangeal joints was evident. There was no arthralgia, arthritis or neurological disorders.

Table 2: Variation in severity of cutaneous manifestations was observed among 10 affected family members studied in detail.

Skin biopsies (Fig. 1) demonstrated mild psoriasiform thickening (acanthosis) of the epidermis, hyperkeratosis, focal and shouldering parakeratosis, scale crusts, follicular hyperkeratotic plugs and overgrowth of Pityrosporum ovale. There were few ectatic blood vessels in the dermis with mild perivascular mononuclear cell infiltrates - of mostly CD4 lymphocytes. However, there was no significant spongiosis typical of SD, and no evidence of clusters of neutrophils in parakeratotic layers as seen in psoriasis (Braun-Falco et al., 1991). Immunohistochemistry demonstrated increased keratinocyte proliferation (Ki67 staining) and upregulation of Keratin 16 in affected skin, as found in psoriasis. Genome-wide linkage analysis of 18 family members (12 affected, 6 unaffected) using 400 microsatellite markers, identified three loci with lod scores higher than 2 (D4S1534, D11S908, D17S928). Fine mapping testing all 54 (19 affected, 35 non-affected) family members available for analysis, ruled out loci D4S1534, Dl 1S908 (results not shown), yet demonstrated linkage to a 0.5 Mb locus at the telomeric end of chromosome 17q25 (Fig. 3) with a maximum multipoint lod score of 8.79 at θ=0 at SNP rs3744165. The entire coding region of all 12 genes and 6 EST's within this locus were sequenced: only a single mutation was identified, in the coding region of ZNFl 50 (Zinc finger protein 750; FLJ13841; NM_024702.1), a gene harboring the SNP rs3744165 within its 5'-UTR. This novel 3 exons gene encodes a protein with a nuclear localization site at its 5' end, and a conserved zinc finger domain (Fig. 4) that starts at amino acid 25. With two histidines and two cysteines that might serve as a zinc binding domain, ZNF750 is a putative member of the C2H2 subclass of zinc finger transcription factors (Pabo et al., 2001). The mutation (56_57dupCC) causes a frameshift resulting in missense coding as of amino acid 19 of this 723 aa protein, leading to a putative 44 aa truncated protein, fully abrogating the zinc finger domain (Fig. 4). Screening with denaturing high-performance liquid chromatography (dHPLC) using sequences of SEQ ID NOs: 98-99 (Fig. 4) showed that all 19 affected individuals tested were heterozygous for the mutation, while none of 35 non-affected family members or 100 non-related individuals of Jewish Moroccan ancestry had the mutation.

To verify the size of the normal ZNFl 50 mRNA, the predicted cDNA segments were amplified by RT-PCR from mRNA prepared from skin biopsies of normal individuals. Comparison of the cDNA sequences to the databases (NCBI, UCSC) indicated that there are no alternative splicing variants. ZNF150 is expressed in the skin, prostate, lungs placenta and thymus, minimally in T cells but not in peripheral blood leukocytes, pancreas and brain. It is clearly expressed in primary human keratinocytes but not in fibroblasts (Fig. 3b).

As seen in Fig. 3d, the mutant transcript is expressed in affected individuals and not in normal controls. Real-time PCR using primer sets specific to the mutant and to the native ZNF750 transcripts (SEQ ID NOs: 8-11) demonstrated that in affected keratinocytes the mutant transcript is not lost to editing. Interestingly, realtime PCR using primers common to the mutant and native ZNF? ⁷50 transcripts, showed that keratinocytes of affected individuals harbor a higher (~2-fold) copy number of ZNF750 transcripts than those of normal controls - perhaps in attempt to compensate for lack of ZNF750 function (Fig. 3f). Members of the zinc finger proteins have been shown to form both homodimers and heterodimers (Pabo et al.,

2001). Therefore, while the mechanism by which abrogation of a single copy of the gene causes disease is yet to be determined, a dominant negative effect is plausible.

ZNF750 is expressed in keratinocytes and not in fibroblasts, suggesting a primary defect in keratinocytes, the major skin cell type affected both in SD and in psoriasis (Plewig et al., 1999; Gupta et al., 2004a; Braun-Falco et al., 1991). As in SD and psoriasis (Plewig et al., 1999; Gupta et al., 2004a; Braun-Falco et al., 1991; Bowcock et al., 2005; Faergemann et al., 2001), infiltrates of CD4 cells, with only minimal CD8 cells, were found in skin biopsies of affected individuals (Fig. 1). While ZNF750 is transcribed in human thymus, it is not expressed in human peripheral blood leukocytes, and its expression in CD4 lymphocytes is minimal, independent of lymphocyte activation (Fig. 3b). Moreover, PMA and Ionomycin- induced T cell proliferation rates of peripheral blood CD4 cells of affected and non- affected individuals were similar (Fig. 3g). Thus, evidence for a primary role of ZNF750 in direct modulation of the immune system in this disorder is limited. However, as keratinocytes secrete cytokines and adhesion molecules (Watanabe et al., 2001), keratinocyte-mediated immunomodulation by ZNF750 is plausible. SD has been associated with a variety of neurological abnormalities, and emotional stress has been shown to aggravate the disease (Plewig et al., 1999; Gupta et al., 2004a). While emotional stress aggravated the skin lesions in affected individuals in this kindred, no neurological symptoms were evident - in line with ZNF750 not being transcribed in the brain. Zinc binding is essential for folding and activity of C2H2 proteins (Pabo et al., 2001). While SD does not respond to supplementary zinc therapy, extreme zinc deficiency in patients with acrodermatitis enteropathica and acrodermatitis enteropathica-like conditions may be accompanied by dermatitis mimicking seborrheic dermatitis of the face (Plewig et al., 1999; Gupta et al., 2004; Braun-Falco et al., 1991). Thus, a possible role for ZNF750 in the seborrheic dermatitis-like presentation of extreme zinc deficiency is yet to be sought. Pityrosporum ovale overgrowth, found in affected individuals here, is thought to play a significant role in SD and has been implicated also in psoriasis (Faergemann et al., 2001). Malassezia yeast species can differentially induce human cytokine production by means of keratinocytes (Watanabe et al., 2001), suggesting a possible mechanism of involvement of Pityrosporum ovale in SD and psoriasis.

Psoriasis is thought to be a complex genetic disease, caused in most cases by the interaction of several common disease alleles genes (Bowcock et al., 2005). Autosomal dominant highly penetrant mostly non-arthritic psoriasis has been associated with the PSORS2 locus (Tomfohrde et al., 1994; Helms et al., 2003; Capon et al., 2005; Hwu et al., 2005; Stuart et al., 2006) harboring ZNF750. The non-arthritic psoriasiform elements in the phenotype described here, suggest that ZNF750 mutations/polymorphisms might underlie variants of psoriasis. SD and psoriasis have many similar clinical and pathological elements (Plewig et al., 1999; Gupta et al., 2004; Braun-Falco et al., 1991). Identification of ZNF750 might open new insights to molecular pathways through which enhanced keratinocyte proliferation, CD4 infiltrates and Pityrosporum ovale overgrowth evolve in those common diseases.

Atopic dermatitis has been previously associated with the 17q25 locus and the PSORS2 locus in that region (Morar et al., 2006; Bowcock et al., 2004; Speckman et al., 2003; Cookson et al., 2001). As we have identified ZNF750 to be the disease-associated gene in the PSORS2 locus, our results heavily suggest association of ZNF750 with atopic dermatitis. EXAMPLE 2.

A mutational analysis screen of genomic DNA samples of 200 "classical" north American psoriasis patients and 50 healthy controls were preformed by overlap PCR amplification of ZNF750 promoter region and gene (SEQ ID NOs: 102-125). Each overlap amplicon was screen for mutation by specific dHPLC program. Two different novel single nucleoide changes (SNCs) were found that were not seen in controls or in any database: These unique mutations are upstream to the ZNF750 gene (-319 C/T) or in the 5^! UTR (+46 CIT), suggesting they might reside in its promoter region. Specifically, in four North American individuals with psoriasis, the following unique changes were found: C/T at position -319 in one patient; C/T at position +46 in three unrelated patients The results are shown in Figs. 5a-c and Figs. 6a-d.

As shown in Fig. 5, a third unique change (A/C position: -347) was found in all affected members (and not in non-affected individuals) in a Taiwanese family presenting with auotsomal dominant inheritance of clear cut psoriasis - a family described in Hwu et al., 2005. This change was not found in 50 Taiwanese healthy controls or in 50 north American healthy controls. While Hwu et al. previously demonstrated that the psoriasis in that family was associated with linkage to the PSORS2 locus, they did not identify the A/G change in position -347 of ZNF750 or any association with the ZNF750 gene.

Luciferase assay: DNA plasmids were generated so that segments from -750 to +78 of the ZNF750 gene sequence from a wild type individual and from a psoriasis patient with the +46C/T mutation were cloned into the pGL3 luciferase reporter plasmid. ρGL3 luciferase reporter plasmid with no promoter served as a negative control. ρGL3 luciferase reporter plasmid carrying the TK mini promoter served as a positive control. These 4 constructs were transiently transfected into HEK293 cell line along with Renilla plasmid that served as an internal control for transfection efficiency. Luciferase activity was tested using standard methodology. As shown in Fig. 7, the +46C/T mutation abrogates expression of the luciferase reporter. Thus, the +46C/T mutation in ZNF750 that was found in three non-related "classical" psoriasis patients affects ZNF750 transcription

EXAMPLE 3.

To identify any further ZNF750 transcripts besides the original one previously described, 5' RACE (Rapid Amplification cDNA Ends) was performed, testing normal human keratinocyte mRNA. In addition to the original transcript, a second, less abundant transcript was found - extending ~500bp beyond the 5' end of the originally described exon 1, encoding a putative 46 aa protein (Fig. 8). All unique SNPs that were found in psoriasis patients are 3' to the stop codon of the second, less abundant transcript, and therefore are less likely to have an effect on this transcript.

APPENDIX

GENECARD FOR THE FLJ13841 GENE TTERMED ALSO ZNF750)

• Hypothetical protein FLJ13841, hypothetical protein LOC79755

. Previous GC identifers: GC17M079921, GC17M084056, GC17M081317, GC17M081466

. Synonyms: MGC 125667, MGC125668

GenelD: 79755 (db_xref)

RefSeq: NM_024702.1 CCDS: CCDSl 1819.1

Entrez Gene: 79755 Stanford SOURCE: NM 024702

Location:

Chromosome 17q25.3

Start: 78,380,600 bp from pter

End: 78,391,188 bp ixompter

Size: 10,588 bases

Orientation: minus strand

mRNA: 78,380,600 - 78,391,140

Coding sequence: 78,381,308 - 78,383,620

Nucleotide Protein mRNA AK023903 BAB14718 mRNA BC109036 AAI09037 mRNA BC109037 AAI09038 5 None Q9H899

The protein:

Size: 723 amino acids; 77387 Da

UniProt/TrEMBL: Q9H899 REFSEQ proteins: NP_078978.1 ENSEMBL proteins: ENSP00000269394

Ontology:

3 Gene Ontology (GO) terms (links to tree view):

GO:0003676 nucleic acid binding GO:0005634 nucleus GO:0008270 zinc ion binding

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Claims

1. A method for diagnosing a disease, disorder or condition of the skin that is associated with the ZNF750 gene (SEQ ID NO. 1) in an individual, comprising determining the identity of a mutation in the ZNF750 gene sequence and/or detecting an aberration in the expression of the ZNF750 gene, in a biological sample of said individual.

2. The method according to claim 1, wherein said aberration in the sequence of the ZNF750 gene is a duplication of the nucleotides CC (56_57dupCC), the mutation -347A/C, the mutation -319C/T or the mutation +46C/T.

3. The method according to claim 1 or 2, wherein the disease, disorder or condition is selected from psoriasiform seborrheic dermatitis, seborrheic dermatitis, atopic dermatitis, acne, tinea versicolor, psoriasis, or any of their variants.

4. A method for identifying a modulator of the ZNF750 gene expression comprising: (i) contacting keratinocytes with a candidate substance; (ii) measuring the transcription of the ZNF750 gene and/or translation of the ZNP750 transcript in the presence or the absence of said candidate substance, wherein a candidate substance capable of modulating the ZNF750 gene transcription and/or translation of the ZNP750 transcript is a modulator of the expression of the ZNF750 gene.

5. A method for identifying a modulator of the activity of a ZNF750 polypeptide (SEQ ID NO: 3) comprising: (i) contacting a ZNF750 polypeptide comprising an amino acid sequence of SEQ ID NO: 3, a fragment or a variant thereof, with a candidate substance; (ii) detecting the complex formed between said polypeptide and said candidate substance; and (iii) measuring the activity of the ZNF750 polypeptide in the presence or the absence of said candidate substance capable of forming a complex with said ZNF750 polypeptide, wherein a candidate substance capable of forming a complex with said ZNF750 polypeptide and changing its activity is a modulator of the activity of said ZNF750 polypeptide.

6. A method for identifying a gene or protein involved in a skin disease, disorder or condition selected from psoriasiform seborrheic dermatitis, psoriasis, seborrheic dermatitis, their variants, acne, Tinea versicolor, atopic dermatitis and/or Pityrosporum infection, said gene or protein being regulated by the ZNF750 gene, comprising: (i) identifying differentially expressed or activated gene(s) or ρrotein(s) in a cell culture exhibiting said disease, disorder or condition characteristics comprising keratinocytes obtained from an individual having said skin disease, disorder or condition and in a cell culture comprising normal keratinocytes; (ii) decreasing or increasing expression of the ZNF750 gene in said cell cultures by contacting the cell cultures with a modulator of claim 4; and (iii) determining the effect of decreasing or increasing expression of the ZNF750 gene on the expression or activity of said differentially expressed or activated gene(s) and/or protein(s) and on the disease characteristics exhibited by the cells of (ii), wherein a change in expression or activity of said differentially expressed or activated gene and/or protein and in the disease characteristics exhibited by said cells is indicative of said differentially expressed or activated gene and/or protein being regulated by the ZNF750 gene and being involved in a skin disease, disorder or condition.

7. The method according to claim 5, wherein the activity of the ZNF750 gene product results in modulation of keratinocyte proliferation or cytokine production by keratinocytes.

8. A method for identifying a gene or protein involved in modulation of Pityrosporum ovale infection, comprising: (i) identification of differentially expressed or activated gene(s) or protein(s) in a cell culture comprising keratinocytes from a sample of skin infected with Pityrosporum ovale exhibiting the characteristics of skin infection and in a cell culture comprising normal keratinocytes; (ii) decreasing or increasing expression or activity of the ZNF750 gene in said cell cultures by contacting the cell cultures with a modulator of claim 4; and (iii) determining the effect of decreasing or increasing expression of the ZNF750 gene on the expression or activity of said differentially expressed or activated gene(s) and/or protein(s) and on the skin infection characteristics exhibited by the cells of (ii), wherein a change in expression or activity of said differentially expressed or activated gene and/or protein and in the skin infection characteristics exhibited by said cells is indicative that said differentially expressed or activated gene and/or protein is regulated by the ZNF750 gene and is involved in modulation of the Pityrosporum ovale infection.

9. A method for treatment of a disease, disorder or condition selected from psoriasiform seborrheic dermatitis, psoriasis, seborrheic dermatitis, their variants, acne, Tinea versicolor, atopic dermatitis and/or Pityrosporum infection, comprising administering to a patient in need a therapeutically effective amount of a molecule selected from: (i) a polynucleotide comprising a nucleotide sequence of the ZNF750 gene, a ZNF750 cDNA (SEQ ID NO: 2), a ZNF750 mRNA, a ZNF750 siRNA, a ZNF750 antisense DNA or RNA, a polynucleotide comprising a nucleotide sequence of a gene homologous to the ZNF750 gene, or a fragment thereof; (ii) a polypeptide comprising a ZNF750 protein, or a fragment thereof; (iii) an antibody specific to a ZNF750 polypeptide, and/or (iv) a modulator of ZNF750 gene transcription and/or translation of the ZNP750 transcript, and/or of ZNF750 protein function.

10. A method for testing the efficacy of a candidate compound for treating a disease, disorder or condition selected from psoriasiform seborrheic dermatitis, psoriasis, seborrheic dermatitis, their variants, acne, Tinea versicolor, atopic dermatitis and/or Pityrosporum infection, comprising contacting keratinocytes exhibiting said disease, disorder or condition characteristics in which the expression of the gene ZNF750 and/or the function of the ZNF750 protein has been partially or completely silenced with a candidate substance, whereby a candidate substance causing reduction in said disease characteristics exhibited by said cells is indicative of the efficacy of said candidate compound in treating said disease, disorder or condition.

11. A method for identifying a compound capable of modulating keratinocyte proliferation and/or cytokine production by keratinocytes, comprising contacting a candidate compound with keratinocytes, in which the expression of the gene ZNF750 and/or the function of the ZNF750 protein has been partially or completely silenced, and comparing keratinocyte proliferation and cytokine production by said keratinocytes with that of normal keratinocytes, wherein a candidate compound capable of modulating keratinocyte proliferation and/or cytokine production by said keratinocytes is a modulator of keratinocyte proliferation and/or cytokine production by keratinocytes.

12. The use of a molecule selected from: (i) a polynucleotide comprising a nucleotide sequence of the ZNF750 gene, a ZNF750 cDNA, a ZNF750 mRNA, a ZNF750 siRNA, a ZNF750 antisense DNA or RNA, a polynucleotide comprising a nucleotide sequence of a gene homologous to the ZNF750 gene, mRNA, siRNA, antisense, homologous gene, or a fragment thereof; (ii) a polypeptide comprising a ZNF750 protein, or a fragment thereof; (iii) an antibody specific to a ZNF750 polypeptide; and/or a modulator of ZNF750 gene transcription and/or translation of the ZNP750 transcript, and/or (iv) a modulator of ZNF750 gene transcription and/or translation of the ZNP750 transcript, and/or of ZNF750 protein function, for the preparation of a pharmaceutical composition for the treatment of a disease, disorder or condition selected from psoriasiform seborrheic dermatitis, psoriasis, seborrheic dermatitis, their variants, acne, Tinea versicolor, atopic dermatitis and/or Pityrosporum infection.

13. A pharmaceutical composition for the treatment of a disease, disorder or condition selected from psoriasiform seborrheic dermatitis, psoriasis, seborrheic dermatitis, their variants, acne, Tinea versicolor, atopic dermatitis and/or Pityrosporum infection, comprising a molecule selected from: (i) a polynucleotide comprising a ZNF750 gene, a ZNF750 cDNA, a ZNF750 mRNA, a ZNF750 siRNA, a ZNF750 antisense DNA or RNA, a gene homologous to the ZNF750, mRNA, siRNA, antisense, homologous gene, or a fragment thereof; (ii) a polypeptide comprising a ZNF750 protein, or a fragment thereof; (iii) an antibody specific to a ZNF750 polypeptide, and a pharmaceutically acceptable carrier; and/or (iv) a modulator of ZNF750 gene transcription and/or translation of the ZNP750 transcript, and/or of ZNF750 protein function.