WO2009053513A1

WO2009053513A1 - Method and kit for determining predisposition to and risk of developing psoriasis and for the diagnosis thereof

Info

Publication number: WO2009053513A1
Application number: PCT/ES2008/070192
Authority: WO
Inventors: Xavier ESTIVILL PALLEJÀ; Rafael Félix DE CID IBEAS; Lluís ARMENGOL DULCET
Original assignee: Fundació Privada Centre De Regulació Genòmica (Crg)
Priority date: 2007-10-24
Filing date: 2008-10-21
Publication date: 2009-04-30

Abstract

The invention relates to a method comprising the following steps: a) isolation of a nucleic acid sample from an individual; and b) analysis of said sample, a predisposition to, risk of developing or diagnosis of psoriasis being indicated if the late cornified envelope gene (LCE3C) is not detected or if a reduction in the number of copies of said gene is detected. The invention also relates to kits for carrying out said method. The method and kit can be used to obtain additional information useful for diagnosis, such that suitable treatment can be devised and the prognosis of said type of disease can be improved.

Description

Procedure and kit for determining predisposition, determining risk to develop or diagnosing psoriasis

The present invention is related to molecular biology. In particular, the present invention relates to a method and kit for determining a predisposition or a risk to develop a dermatological disease, in particular psoriasis.

STATE OF THE PREVIOUS TECHNIQUE

Psoriasis is a chronic disease of the skin that occurs in different clinical forms: plaques, "guttata", erythrodermic and pustular. Although psoriasis affects all ethnic groups, its prevalence varies depending on the geographic location. The prevalence in northern Europe is close to 3%, in North America and in the United Kingdom it is 2%, while in Japan it is 0.2%, being rare in Native Americans. The concordance of psoriasis in monozygotic twins is 35-90%, compared to 12-30% in dizygotic twins, with an estimated heritability of 80-90%. Psoriasis affects both men and women and the first manifestation of the disease usually occurs in the third decade of life.

Psoriasis affects millions of people worldwide and does not have a curative treatment or there are no biological methods for its diagnosis, which is based on exclusively clinical criteria. On the other hand, there is no clear treatment modality for all individuals suffering from psoriasis.

An early prescription of an effective drug treatment is critical, as this would avoid the risk of progressive deterioration of

Ia skin, reaching situations of chronicity, superinfections of the skin or other disorders, which can affect the joints (psoriatic arthritis). On the other hand, psoriasis can lead to significant aesthetic disorders in the person, which may have psychological consequences for the individual suffering from the disease. The location of genes possibly involved in dermatological pathologies such as psoriasis can be complex due to the imprecise definition of phenotypes, the heterogeneity and the uncertainty of the genetic transmission of the disease trait. It is considered that a diagnosis before the symptoms of the disease can emerge could prevent its occurrence or lessen its consequences.

On the other hand, the biology of psoriasis is still poorly understood. It is believed that the disease is due to the combination of genetic and environmental factors, including skin lesions, infections, stress and medications. Alterations of the skin of patients with psoriasis include accelerated maturation and hyperproliferation of keratinocytes and inflammation with an increase in the presence in the skin of T lymphocytes.

In an attempt to elucidate the genetic component of psoriasis, different genetic studies have been carried out in order to locate chromosomal regions that play a decisive role in psoriasis susceptibility. Studies have been conducted in families with several affected psoriasis members, thus identifying several chromosomal regions. Since the effect of these regions has been restricted to few family nuclei, additional studies have been carried out thus defining loci or chromosomal locations for psoriasis. Among them, it is worth highlighting the major histocompatibility complex (HLA), of which its involvement in psoriasis has been demonstrated in association studies using cases and controls. This region of the HLA corresponds to HLA-Cw6 and the locus in which it is found is known as PSOR1. Loci not linked to HLA have been detected on chromosomes 17q25 (PSORS2), 4q35 (PSORS3), 1q21 (PSORS4) and 3q21 (PSORS5). Despite these advances, none of the genes involved in this disease have yet been identified.

On the other hand, associations between psoriasis and other processes such as atopic dermatitis have been detected. Thus, for example, in recent publications such as Giardina E. et al., (Cf. Giardina E. et al., "Co-localization of susceptibility loci for psoriasis (PSORS4) and atopic dermatitis (ATOD2) on human chromosome 1 q21 ", Human Herediary, 2006, vol. 61, p. 229-236) se indicates that the 1 q21 chromosomal region predisposes both psoriasis and atopic dermatitis.

Despite the efforts made to date to improve the diagnosis of patients with psoriasis, it is not possible to identify early the possible predisposition or susceptibility of an individual to develop the disease.

EXPLANATION OF THE INVENTION

The present invention is based on the finding that a gene belonging to the cluster of late differentiation genes of the epidermal stratum corneum (in English "late cornified envelope", abbreviated as "LCE") is related to the predisposition or risk to which an individual develop psoriasis In particular, the inventors have verified in nucleic acid samples of individuals who have already developed psoriasis that the late differentiation gene of the epidermal stratum corneum 3C (hereinafter abbreviated as LCE3C) is not found or presented in a decreased number of copies regarding samples of control individuals without the disease.

Thus, in a first aspect the present invention refers to a procedure for the determination of a predisposition, a risk to be developed or the diagnosis of psoriasis, said procedure comprising: a) isolating a sample of nucleic acid from an individual; and b) analyze said sample, so that if the late differentiation gene of the epidermal stratum corneum 3C (LCE3C) is not detected or a decrease in the number of copies thereof is detected, it is indicative of a predisposition, risk to develop or Psoriasis diagnosis.

In fact, as illustrated below, researchers have seen that in patients with psoriasis a partial or complete deletion of at least one copy of the late differentiation gene of the epidermal stratum corneum 3C (LCE3C) takes place, which gives rise to to the non-detection of the gene (absence) or to a decrease in the number of copies thereof regarding a control. Therefore, the partial or total deletion of at least one copy of LCE3C confers risk to develop or appearance of psoriasis. The late differentiation proteins of the epidermal stratum corneum are encoded by 13 genes located in the epidermal differentiation complex of chromosome 1 q21 (PSOR4) in a region of about two megabases. These genes have been classified into three groups and there are also several pseudogenes thereof. Of the three groups, group 3 contains five genes (LCE3A, LCE3B, LCE3AC, LCE3D and LCE3E) (see FIG. 1). The expression of the LCE genes is induced by calcium and ultraviolet irradiation and the LCE proteins are part of the structural component of the corneal layer of the skin. The cDNA sequences encoding these genes are available in public databases such as GenBank, where to access the cDNA sequence of LCE3A the accession number is NM_178431, of LCE3B is NM_178433, of LCE3C is NM_178434, of LCE3D is NM_032563 and of LCE3E is NM_178435). The genes that code for LCE are located on chromosome 1, specifically in region 1 q21.3. In said chromosome the genes that code for group 3 are located between positions 150804754 (LCE3E) and 150862203 (LCE3A), according to the assembled sequence of March 2006 (hg18) (http://genome.ucsc.edu/cgi- bin / hgGateway). In Jackson et al. (Jackson, B. et al., "Late cornified envelope family in differentiating epithelia -response to calcium and ultraviolet irradiation" 2005, Journal Investigative Dermatology vol. 124, p. 1062-1070) provides information on the cluster of LCE genes, its expression profiles and the response to stimuli such as ultraviolet radiation.

To date, scientific publications (both literature and patent applications) had described the involvement of region 1q21 in psoriasis in family cases. The colocalization of psoriasis and atopy in said chromosomal region has also been defined (Giardina, E. et al., (Supra)). In Jackson et al., (Supra) the existence of heterogeneity was indicated among individuals with a loss of the individual expression of a gene of the LCE cluster without manifesting a skin disease, suggesting, in turn, that the genes of the Cluster of LCE genes subtly affect skin functions. In addition, after carrying out a study of heterogeneity of LCE in the population, they concluded that the results obtained indicated that the ablation of the expression of a single gene of the LEC cluster had minimal effects on the function of the skin. Surprisingly, the inventors of the present invention have found that the total or partial deletion of at least one copy of the LCE3C gene is decisive in the predisposition and susceptibility to develop psoriasis. The results obtained (as illustrated in the examples below) indicate that LCE3C can be used to determine predisposition or susceptibility with high precision as well as provide useful information for the diagnosis of psoriasis.

In a second aspect, the present invention provides kits for carrying out the step (b) of the process according to the first aspect of the invention.

Unless defined otherwise, all technical and scientific terms used in this document have the same meaning as the one usually understood by the person skilled in the art to which the invention belongs. Methods and materials similar or equivalent to those described herein can be used in the practice of the invention. Throughout the description and the claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the LEC gene cluster, according to the publication of Jackson et al. The boxes show the intron-exon structures and the arrows indicate the direction of the transcription. The exons of LCE3C are indicated separately (E1 -E3). The numbers represent the distance between genes in kilobases.

DETAILED EXHIBITION OF REALIZATION MODES

In the genome of an individual there may be repeated regions that include genes from the LEC cluster and, in particular from the LCE3 sub-cluster. In fact, on chromosome 1 (where the LCE cluster is located) there may be a single copy of the LCE3C gene or several copies thereof.

Thus, the expression "at least one copy of the late differentiation gene of the epidermal stratum stratum 3C (LCE3C) is partially or completely deleted" means that when performing step (b) of the procedure or the LCE3C gene is not detected ( Which means that the only copy of said gene is deleted in the individual's sample) or a decrease in the number of copies of the gene is detected as a result of the total or partial deletion of one or more copies thereof with respect to controls well established (as illustrated below).

In a preferred embodiment of the first aspect of the invention, when stage (b) is carried out, the late differentiation gene of the epidermal stratum corneum (LCE3C) is not detected in the patient sample.

At the time of determining in a sample of a patient the absence of the LCE3C gene or the decrease in the number of copies different methodologies are carried out but at the same time complementary. In accordance with preferred embodiments of the process of the first aspect of the invention, certain single nucleotide polymorphisms can be genotyped, which provide information on the total loss of LCE3C. Thus, when the genotype of certain single nucleotide polymorphisms is analyzed (as mentioned below) the genotype obtained will indicate whether or not there are copies of the gene. In the event that the genotype is indicative that there is no copy of LCE3C, this will be indicative in turn that there has been complete deletion of the copy (s) of the LCE3C gene and consequently, that there is a predisposition or risk to develop psoriasis If the genotype obtained through the analysis of the polymorphisms is not conclusive (that is, it does not allow to determine that there is a complete absence of LCE3C), the number of copies present in the patient sample is determined and compared with the number of copies of a specific control. If it is observed that the number of copies of the LCE3C gene in the patient is less than the number of copies of the control, it will imply that one or more copies of the LCE3C gene in the individual's sample have been deleted and, consequently, there is a predisposition to develop psoriasis. . The term "totally or partially deleted" means that the entire gene is deleted or a fragment thereof (eg, an exon from LCE3C).

When the expression "absence" relative to the LCE3C gene is used in the present invention, it means that when stage (b) is carried out in accordance with the procedure of the first aspect of the invention, no copy of the LCE3C gene is detected in Ia Sample of the individual.

In an embodiment of the first aspect of the invention, step (b) is carried out by analyzing at least one single nucleotide polymorphism in the cluster of late differentiation genes of the epidermal stratum corneum. These polymorphisms can be located in any of the genes that are part of said cluster as well as in the intergenic regions between these genes.

In the present invention, "single nucleotide polymorphism" or SNP means a region of a gene, or regulatory elements thereof, where the sequence of nucleotide bases can vary between individuals in a given nucleotide. There is often a predominant genotype that represents the usual form of a gene with subsets of a population that has a polymorphism that confers a different genotype. Certain polymorphisms may prevail in individuals of a particular ethnicity or specific geographic areas. A polymorphism may not affect the function of the gene, may lead to differences in the function of the gene, may produce an inactive product of the gene or may modulate the production of the product of the gene.

Preferably, said polymorphism is selected from Table 1:

The polymorphisms that are part of the object of the invention are well known in the state of the art and additional information about them can be obtained in different public databases such as the NCBI (http: //www.ncbi.nlm.nih. gov) or Ia UCSC (http://www.genome.ucsc.edu). The reference number (rs #) referred to in the present invention for each of the polymorphisms is that assigned by the NCBI and will be the form used hereafter to refer to them.

Preferably, in step (b) the polymorphism of a single nucleotide selected from the group consisting of: rs17659359, rs17659389, rs7516108, rs10888502, rs12046030, rs4112788 or combinations thereof are analyzed. More preferably single nucleotide polymorphisms are selected from rs4112788, rs17659359 and rs12046030.

In a preferred embodiment of the first aspect of the invention, step (b) is carried out by genotyping a single nucleotide polymorphism, so that if the genotype resulting from the analysis of the single nucleotide polymorphism rs4112788 or rs10888502, is homozygous CC, is indicative of the absence of the LCE3C gene (that is, none of said gene is detected in the individual's sample) and consequently, indicative that there is a predisposition for the individual to develop psoriasis. When the procedure object of the invention is carried out analyzing the polymorphisms located in the LCE3D and LCE3E genes, the CC genotype shows the highest degree of association with the absence of the LCE3C gene. The association of the C allele of rs4112788 with the absence of LCE3C is such that 97.5% of the homozygous subjects for the C allele do not have the LCE3C gene. This association has a statistical value of 3.14 E-85 (Table 3 below), which is indicative of the accuracy of the information that can be obtained when determining the predisposition or susceptibility to develop psoriasis. On the other hand, none of the subjects homozygous for the T allele are homozygous for the absence of the LCE3C gene. This allows the use of SNP rs4112788 (and also others or their combinations) as a marker of the deletion of LCE3C, and consequently for the determination of a predisposition or risk to develop or diagnosis of psoriasis.

In the described methods, the alleles present in a sample are identified by determining the nucleotide present in one or more of the polymorphic points. A series of methods for the identification of nucleotides present in polymorphic sites are known in the art.

The particular method used to identify the genotype is not a critical aspect of the invention. Although for considerations of realization, cost and convenience some particular methods will be more desirable than others, it is clear that any method that can identify the present nucleotide will provide the necessary information to identify the genotype.

Preferred genotyping methods involve DNA sequencing, allele specific amplification or detection based on amplified nucleic acid probes.

The alleles of LCE3C and the cluster of LCE genes can be identified by DNA sequencing methods, such as the method of termination of the chain (Sanger et al., 1977, Proc. Nati. Acad Sci., 74: 5463-5467), which is well known in the field. In one embodiment, a sub sequence of the gene that includes the polymorphic site is amplified and cloned into a suitable plasmid and then sequenced, or sequenced directly. Based sequencing the PCR described in patent US 5,075,216 N ^0. Normally, sequencing is performed using one of the Automatic DNA sequencers that are commercially available, including new generation based on DNA synthesis or DNA ligation.

The alleles of LCE3C and the other polymorphisms of the cluster of LCE genes can also be identified using genotyping methods based on amplification. Several methods of amplification of nucleic acids known in the art can be used to detect nucleotide changes in a target nucleic acid. A preferred method is the reaction in polymerase chain (PCR), which is now well known in the art, and described in US Patents 4,683,195 ^0, 4,683,202 and 4,965,188. Using the genotyping based on the specific allele amplification, the identification of the alleles only requires the detection of the presence or absence of the amplified target sequences. The methods for the detection of the amplified target sequences are well known in the art. Genotyping methods based on allele specific amplification can facilitate the identification of haplotypes, as described in the examples. Essentially, the allele specific amplification is used to amplify a region that includes multiple polymorphic sites of only one of the two alleles in a heterozygous sample.

The variants of the SNP present in the amplified sequence are then identified, by means of the hybridization of probes or sequencing.

Probe - based genotyping can be performed using a test or "TaqMan""5'nuclease" as described in US Patent 5,210,015 N ^0, 5,487,972 and 5,804,375. In the TaqMan assay, labeled detection probes are added that hybridize with the amplified region in the amplification reaction mixture. The probes are modified to prevent the probes from acting as primers for DNA synthesis. Amplification is performed using a DNA polymerase that has 5 'to 3' exonuclease activity. During each step of synthesis of the amplification, any probe that hybridizes with the target nucleic acid following the primer to be extended is degraded by the 5 'to 3' exonuclease activity of the DNA polymerase. Therefore, the synthesis of a new target chain also results in the degradation of a probe, and the accumulation of the degradation product provides a measure of the synthesis of target sequences.

Any suitable method for the detection of the degradation product can be used in the TaqMan test.

The TaqMan assay can be used with allele-specific amplification primers so that the probe is used only to detect the presence of amplified product. Such an assay is performed as described for the kinetic PCR described above. Alternatively, the TaqMan assay can be used with a specific probe of the target. The assay formats described above normally use labeled oligonucleotides to facilitate the detection of hybrid duplexes. The oligonucleotides can be labeled by the incorporation of a signal detectable by spectroscopic, photochemical, biochemical, immunochemical, radiological, radiochemical or chemical methods. Useful tides include ³² P, fluorescent dyes, electrodensactive reagents, enzymes (as normally used in ELISAs), biotin or haptens and proteins for which antisera or monoclonal antibodies are available. The labeled oligonucleotides of the invention can be synthesized and labeled using the techniques described above for oligonucleotide synthesis. After hybridization of the target DNA immobilized with the biotinylated probes under specific sequence conditions, the probes that remain attached are detected by a first binding to the biotin of horseradish avidin-peroxidase (AHRP) or horseradish streptavidin-peroxidase (SA -HRP), which is then detected by carrying out a reaction in which the HRP catalyzes a color change of a chromogen.

In accordance with the present invention, the polymorphisms associated with psoriasis are analyzed. Using the information on the polymorphisms obtained can determine the risk of developing psoriasis with high precision. Therefore, the present invention is an effective means of knowing the risk of developing psoriasis before symptoms appear. In addition, according to the present invention, an auxiliary information useful for diagnosis is obtained, so that a suitable treatment can be designed and the prognosis can be improved. In addition, Ia The present invention provides useful information in the clarification of the mechanism of development of psoriasis and is expected to contribute to the prevention and treatment of said disorders. In the same way, it is expected that the present invention can contribute to the development of new drugs that allow the treatment of psoriasis based on the presence of the polymorphisms indicated above.

In another preferred embodiment of the first aspect of the invention, step (b) is carried out by determining the number of copies of the LCE3C gene in the individual's sample, and comparing the resulting number of copies with the number of copies of the LCE3C gene identified. in a control sample.

The control sample has been defined from the study of the number of copies of the LCE3C gene in samples belonging to the public HapMap project (http // www.hapmap.org). The samples included in the study to determine the number of copies as controls have been; NA18515, NA18516, NA18517, NA19127, NA19128, NA19129. All samples of the HapMap project have been studied by different methodologies regarding the presence of variations in the number of copies, and all the information is publicly accessible in the public database,

Datábase of Genomic Variation (http://projects.tcag.ca/variation/project.html).

In the present invention, "variation of copy number" (hereinafter referred to as "CNV") means a change in the number of copies due to the duplication or deletion of an amount of DNA greater than one kilobase (see Freeman et al., "Copy number variation: New insights in genome diversity", Genome Res., 2006, vol. 16, p. 949-961). The existence of repeated genome regions that include genes from the LEC cluster and, in particular from the LCE3 sub-cluster, is known in the state of the art. It should also be noted that these CNV regions that include LCE3 sub-cluster genes have not been associated with any known pathology. There are public databases in which information is provided on the variations of copy number that include genes of the LCE3 sub-cluster. For example, in the database of genomic variants (http://projects.tcag.ca/variation) the following information regarding the variations of copy number associated with the LCE3 sub-cluster is found, but no information is provided regarding to your relationship with illness. According to the above, on chromosome 1 (where the LCE cluster is located) there may be several copies of LCE3C and said individual does not develop psoriasis. From the data obtained it is concluded that the decrease in the number of copies of the LCE3C gene (due to the deletion of said gene) or of other genes of the cluster will be indicative of predisposition or susceptibility to develop psoriasis. Although there is a predominant genotype with respect to the CNV in question that represents the usual genomic form with subsets of a population that has a CNV that has a different genotype, certain CNVs may prevail in individuals of a certain ethnicity or specific geographic areas. In this way, a CNV can have consequences regarding the expression levels of a gene directly or indirectly, being able to affect the function of the gene, produce an inactive product thereof or modulate the production of the product of a gene that is found in the neighborhood. These are the relationships established for the CNV of the LCE cluster and the predisposition or protection for the development of psoriasis.

The genotyping of CNVs is carried out in the alleles of the DNA sample, identifying the genomic region that defines the CNV of the LCE cluster (using in part the information available in the databases since this is still very inaccurate in terms of number of copies that the different subjects have). Methods of identifying CNVs that include comparative genomic hybridization or molecular quantitative methods are well known in the state of the art. For a review of the methods used, Estivill and Armengol can be seen (cf. Estivill X., Armengol L. "Copy number variants and common disorders: Filling the gaps and exploring complexity in genome-wide association studies" PIoS Genetics, 2007 , in press) or Scherer et al. (cf. Scherer S. et al. "Challenges and standards in integrating surveys of structural variation" Nature Genetics, 2007, vol. 39, pS7-S15). The procedure used to carry out the genotyping of CNVs is not a critical aspect of the invention.

Several of the SNPs that are part of the object of the invention allow the CNVs of the LCE region to be detected, being preferentially associated with the absence of the LCE3C gene or with a decrease in the number of copies of the gene. Table 5a (below) describes these SNPs and their degree of association with the absence in homozygosis of the LCE3C gene.

In a preferred embodiment of the invention, the samples of the patients with the different clinical forms of psoriasis are selected to make the prediction or diagnosis based on the procedure according to the invention. This depends on the association between psoriasis and the specific SNP or CNV. These associations were established by the inventors by carrying out additional experiments and statistical analyzes, as explained below.

The proportion of data based on the association analysis allows the specialist to interpret the importance of the genotypes identified by sequencing the patient's DNA. In this way, the medical specialist can assess the probability that a patient has or develops psoriasis. It will be evident that the data relating the association of a particular genotype with a disorder can be provided to the user of the method according to the invention by incorporating an information sheet as part of a kit. Thus, in a second aspect the present invention provides a kit comprising suitable means for carrying out the step (b) of the process according to the first aspect of the invention.

The kit may contain specific oligonucleotide probes for each of the polymorphisms, whether SNPs or CNVs, as well as instructions for their use. In some cases the detection probes may be fixed on a suitable support membrane. The kit can also comprise amplification probes to amplify a region in which the polymorphism is found, such as primers. Alternatively, the kit may contain a set of primers comprising an allele-specific probe for the specific amplification of the alleles. The kit may contain the reagents necessary to sequence the region containing the psoriasis-associated SNPs in the cluster of the LCE genes and the CNVs that contain them. These reagents may include those necessary for Sanger type sequencing, synthesis or ligation. Other optional components of the kit include additional reagents used in genotyping methods of SNPs or CNVs well known to those skilled in the art. For example, the kit can additionally comprise an agent that catalyzes the synthesis of primer extension products, reagents for the marking and / or detection of nucleic acid and buffers suitable for the amplification or hybridization reaction.

Preferably, the kit comprises suitable means for carrying out the analysis of the single nucleotide polymorphisms of Table 1 or of the variation sequences of the copy number of Table 2. More preferably, suitable means comprise one or more oligonucleotides of specific sequence, each of the oligonucleotides comprising a sequence that hybridizes under strict conditions with at least one of the polymorphisms of Table 1 or with at least one of the variation sequences of the copy number of Table 2. Even more preferably , said suitable means comprise one or more oligonucleotides of specific sequence, each of the oligonucleotides comprising a sequence that hybridizes under strict conditions with the variable regions in copy number or CNVs or with nucleotide polymorphisms or SNPs. EXAMPLES

SUBJECTS OF STUDY

196 subjects with psoriasis from different clinical centers in Barcelona were recruited. In the study, 232 healthy volunteer subjects recruited from the blood bank of the Hebron VaII Hospital in Barcelona were used as samples. In all cases the permits to the Research Committees were obtained and informed consent was obtained from the patients and controls.

A. ANALYSIS OF SNPs

A.1 Selection of SNPs in the LCE gene cluster

SNPs were selected from the public database dbSNP version db124 (http://www.ncbi.nlm.nih.gov/) by placing LCE the search engine and selecting SNPs in humans. In non-synonymous coding SNPs, preference was given to those having an allelic frequency greater than 5% in European populations or greater than 10% in other populations. In the case of not having allele frequencies, those validated by multiple independent submissions. The SNPs from the HapMap public database (http://www.hapmap.org), data from phase 1 (June 2005), assembly B34 of NCBI and dbSNP124 were selected. First, those SNPs with a frequency greater than 10% were selected in the European sample (CEU) and located in an LCE region. If there were more than two SNPs in high linkage imbalance (r ² > 0.95) only one of them was selected. Those with an allelic frequency greater than 10% in the European population or greater than 30% in other populations were considered good candidates. The initial selection was 284 SNPs in a region of about 200 kb that contains the LCE genes. The final result was the selection of 32 tagSNPs that were genotyped in the total patient and control samples.

SNPs derived from this process and that have shown positive association in the study for LCEs are shown in Table 1 above. A.2. Genotyping of polymorphisms

All SNPs were genotyped using the SNPlex system (Applied Biosystems). The SNPs were launched to design using the tool provided by Applied Biosystems on their website

(https: // ms. appliedbiosystems.com). The oligonucleotides for genotyping were designed and manufactured by Applied Biosystems (Applied Biosystems, Foster City, CA). For genotyping, a slightly modified protocol was originally followed by the one originally indicated by the manufacturer (Tobler AR, et al., "The SNPlex genotyping system: a flexible and scalable platform for SNP genotyping", J Biomol Tech, 2005, vol. 16 (4 ), p. 398-406). All reactions were done in 384-well plates using several liquid handling robots. Specifically, 12 ul of each DNA sample to 75 ng / .mu.l boiling fragmented to 99 ⁰ C for 10 minutes. Two microliths (150 ng) were dehydrated in a 384 well plate. The ligation reaction was carried out, for each well, in a total volume of 5 μl containing 2.3 μl of water without nucleases, 2.5 μl of oligonucleotide mixture, 0.05 μl of universal linkers, 0.1 μl of probes and 0.05 μl of dATP (100x). The PCR reaction consisted of an initial heating at 48 ⁰ C of 30 minutes, initial denaturation at 9O ⁰ C for 20 minutes, 25 cycles of denaturation at 94 ⁰ C for 15 seconds, hybridization at 6O ⁰ C for 30 seconds and extension to 51 ⁰ C for 30 seconds (with 3% increase), final extension at 99 ⁰ C for 10 minutes and maintenance at 4 ⁰ C until the next step. Washing of the PCR with exonuclease was carried out, for each well, in a final volume of 5 μl containing 4.2 μl of water without nucleases, 0.5 μl of buffer of the exonuclease, 0.2 μl of lambda exonuclease and 0.1 .mu.l of exonuclease I. the plates were kept in a thermocycler for 90 minutes at 37 ⁰ C, followed by 10 minutes at 8O ⁰ C and holding at 4 ⁰ C until the next step. The final amplification reaction was carried out, for each well, in a final volume of 7.92 μl containing 2.42 μl of water without nucleases, 5 μl of SNPLexAMp Mastermix and 0.5 μl of oligonucleotides (2Ox). The PCR reaction consisted of an initial denaturation at 95 ⁰ C for 10 minutes, 30 cycles of denaturation at 95 ⁰ C for 15 seconds, hybridization at 63 ⁰ C for 1 minute and maintenance at 4 ⁰ C until the next step. Finally, each of the specific fluorescent probes were eluted and analyzed in an Applied Biosystems 373OxI DNA analyzer Analyzer The GeneMapper software v4 program (ABI, Foster City, CA) was used to analyze the raw data and assign the individual genotypes.

A.3. Statistical methods

The case-control association tests were carried out by logistic regression analysis. It was assumed that the most frequent allele in the controls was the common allele and the homozygous genotype for this was considered as the reference category. Ratios Odds (OR) and 95% confidence intervals (95% Cl) for each genotype were calculated by comparing them with the reference category. Three genetic models (dominant, recessive and additive) were considered. For each polymorphism, P values were computed using the likelihood test. All analyzes were carried out using the SNPassoc statistical package (González, J. R. et al. "SNPassoc: an R package to perform whole genome association studies",

Bioinformatics, 2007, vol. 23, p. 644-645 ") and haploview analysis software (Barret JC et al.," Haploview: analysis and visualization of LD and haplotype maps ", Bioinformatics, 2005, vol. 21 (2), p. 263-265). , the results summarized in Tables 1, (from above), and Tables 3, 3a and 4, 4a were reached:

From the results presented in Table 3, it follows that individuals with the CC genotype for SNP rs4112788, are carriers of the homozygous deletion of the gene. The significance, or certainty of this statement is contained in the value of P, which is much lower than the value of 0.05, established this, as the margin of error due to chance. This value of p = 1.62 E-85 obtained in our experiment, indicates that our result should not be considered as a false positive, obtained by chance conditions. From the results shown in Table 4, it follows that individuals with the CC genotype for SNP rs4112788 have a higher risk of psoriasis than individuals who are heterozygous (CT) or homozygous for the other allele (TT). The significance, or certainty of this statement is contained in the value of P, which is less than the value of 0.05, established this, as the margin of error due to chance. This value of p = 0.0099 obtained in our experiment, indicates that our result should not be considered as a false positive, obtained by chance conditions.

From the results presented in Table 3a and 4a, it is derived that variations in other SNPs of the LCE cluster have similar implications regarding defining genotypes of increased risk of psoriasis (Table 3a), or serving as indirect markers of the presence of the deletion in homozigosis of the LCE3C gene (table 4a). In tables 3a and 4a, more extensive, it is not detailed The genotype of each marker, but the information of the association is contained in the value of P, which is lower than the value of 0.05, established this, as the margin of error due to chance. This value of p <0.05 obtained in each experiment, indicates that the result should not be considered as a false positive, obtained by chance conditions.

Specifically from the results for SNP rs4112788, it follows that the CC genotype can be used as an indirect marker of the presence of the deletion in homozigosis of the LCE3C gene, and that on the other hand individuals carrying the CC genotype have an increased risk of suffer from psoriasis, and can be used as a risk marker for the disease.

B. DETERMINATION OF VARIATION IN THE NUMBER OF COPIES OF LCE3C

B.1. Identification of a region of chromosome 1 q21 that shows variation of the copy number in patients with psoriasis

The identification of the region of the LCE3C gene involved in psoriasis was identified based on comparative genomic hybridization (CGH) experiments using the Agilent 244K array (following the manufacturer's instructions). The analysis identified a region of 4 contiguous probes that showed a decrease in the intensity of the hybridization (Odds Ratio> 0.3) in the psoriasis samples with respect to the control samples. The region involves Agilent probes A_16_P15296679, A_14_P100633,

A_16_P15296703 and A_16_P00165273. These probes include the LCE3C gene. Three TaqMan probes (two flanking and one from the region of the LCE3C gene) were used to check for changes in the number of copies between patient samples and control samples (Table 5): Table 5. Markers for the characterization of the epidermal differentiation complex

* commercial probe located in the exonic region of the NM_178429.2 gene, Applied Biosystem, Foster City, CA, USA.

** commercial probe located in the third exon region of the NM_178434.1 gene *** commercial probe located in the exonic region of the NM_178435.2 TaqMan gene,

TaqMan® Gene Expression Assays (Applied Byosistems) Agilent, Agilent 244K array probes that show dose variability

A probe for LCE2C and another for LCE3E in Table 5 were selected in order to confirm that said genes were not affected (control) and, consequently, to rule out their possible involvement in the disease. In an analysis of 195 cases of psoriasis and 232 controls, the frequency of completely or partially deleted samples was compared with respect to those with one or more copies of the LCE3C gene. The study found that only the probe of the LCE3C gene had variation in the gene dose. Table 6 shows the results of the study with the TaqMan probe of LCE3C.

From the results presented in Table 6, it is derived that individuals with fewer copies of the LCE3C gene (RQ <0.5), have a risk 1.49 times higher (Odds Ratio = 1.49) of suffering from psoriasis. The significance, or certainty of this statement is contained in the value of P, which is less than value of 0.05, established this, as the margin of error due to chance. This value of p = 0.04 indicates that our result should not be considered as a false positive, obtained by chance conditions.

B.2. Effect of genotypes of the polymorphisms of the LCE region on the expression of the genes of the LCE cluster

To evaluate the functional role of the CNV of LCE and SNPs that are associated with it and psoriasis (rs17659389, rs7516108, rs10888502, rs12046030, rs4112788, rs12034595, rs4845448, rs4845456, rs11205050, rs1332244, rs1332244, rs1332244, rs1332244, rs1332244, rs1332244, rs1332244, rs1332244, rs1332244, rs1332244, rs1332244, rs1332244, rs1332244, rs1332244, rs1332244, rs1332244, rs1332244, rs1332244 information on the expression values of the LCE5A, LCE3E, LCE3D, LCE3C, LCE3B, LCE3A, LCE2D, LCE2C, LCE2B and LCE2A genes of the HapMap lymphoblast lines (Stranger BE et al. "Relative impact of nucleotide and copy number variation on gene expression phenotypes ", 2007, Science, vol. 315, pp. 848-853) corresponding to Illumina probes GI_30410034.S, hmm25905-S, hmm25906-S, GM4211868-S, GL30410036-S, GI_30410040-S, GI_30410038 -S, GI_41106713.S, GI_30410042.S, GI_30410046.S, GI_21614551.S, and GI_30410044.S. We evaluated the correlation of the different genotypes of the SNPs (Stranger BE et al., Supra) and found that the genotype correlated with significantly different levels of expression (see Table 7), which would be in accordance with a positional effect in relation to Ia association with psoriasis This correlation opens experimental lines on the genes of the LCE cluster that participate in the development of psoriasis, which could, in turn, have implications at the therapeutic level.

From the results presented in Table 7, it follows that the nucleotide variations evaluated in our study, in the genes or adjacent regions of the genes of the LCE cluster, affect the expression of the various genes of the cluster. In Table 7, the significance, or certainty of this statement is contained in the value of P shown in the table (for example, SNP rs12046130 affects the expression of LCE5A, value of p = 0.0024). The values of p lower than the value of 0.05, established this as the margin of error due to chance, indicates that these variations are not neutral in terms of the functional implication on the genes of the LCE cluster.

The results obtained suggest that the procedure for determining predisposition, risk to develop or diagnosis of psoriasis, according to the present invention, based on the detection of LCE3C, may offer additional information if combined with the detection of a variation in the number of copies of one or more genes contained in the LCE cluster such as LCE3B, LCE3A, LCE5A, etc.

Claims

1. Procedure for determining a predisposition, risk to develop or diagnosis of psoriasis, said procedure comprising: a) isolating a sample of nucleic acid from an individual; and b) analyze said sample, so that if the late differentiation gene of the epidermal stratum corneum 3C (LCE3C) is not detected or a decrease in the number of copies thereof is detected, it is indicative of a predisposition, risk to develop or Psoriasis diagnosis.

2. Method according to claim 1, wherein in step (b) the LCE3C gene is not detected.

3. Method according to claim 1 or 2, wherein step (b) is carried out by analyzing at least one single nucleotide polymorphism in the cluster of late differentiation genes of the epidermal stratum corneum.

4. Method according to any of claims 1 -3, wherein the single nucleotide polymorphism is selected from those shown in Table 1.

5. Method according to claim 4, wherein the nucleotide polymorphism is selected from the group consisting of: rs17659359, rs17659389, rs7516108, rs10888502, rs12046030, rs4112788 and combinations thereof.

6. Method according to claim 5, wherein the single nucleotide polymorphisms are selected from rs4112788, rs17659389 and rs12046030.

7. Method according to claim 6, wherein the analyzed polymorphism is r4112788 or rs10888502 and the homozygous CC genotype is indicative of the absence of the LCE3C gene.

8. Method according to claim 1, wherein the step (b) is carried out by determining the number of copies of the LCE3C gene in the sample, and comparing the resulting number of copies with the number of copies of the gene LCE3C identified in a control sample.

9. Kit for carrying out the procedure defined in claim 1 comprising suitable means for carrying out the analysis of step (b).

10. Kit according to claim 9, comprising suitable means for carrying out the analysis of the polymorphisms shown in Table 1.

11. Kit according to claim 9 comprising suitable means for carrying out the analysis of the variations in the number of copies of the Ia

Table 2.

12. Kit according to any of claims 9 to 11, wherein suitable means comprise one or more oligonucleotides of specific sequence, each of the oligonucleotides comprising a sequence that hybridizes under strict conditions with at least one of the polymorphisms of Table 1 or with at least one of the sequences variation of the copy number of Table 2.

13. Kit according to claim 12, wherein the suitable means comprise one or more oligonucleotides of specific sequence, each of the oligonucleotides comprising a sequence that hybridizes under strict conditions with the polymorphisms rs4112788, rs17659389 and rs12046030.

14. Use of a kit according to any of claims 9 to 13 for the determination of a predisposition, risk to develop or the diagnosis of psoriasis.

15. Use of single nucleotide polymorphism rs4112788, rs17659389 or rs12046030 for the determination of a predisposition, risk to develop or diagnosis of psoriasis.