WO2009093288A1 - Marker gene for examining psoriasis vulgaris - Google Patents

Abstract

It is intended to identify a novel psoriasis vulgaris-sensitive gene by a mapping method using microsatellites at a high efficiency and a low cost. Namely, a novel psoriasis vulgaris-sensitive gene such as OR2J3 gene or IGSF4D gene in human genomic DNA sequence has been identified by conducting a case control correlation analysis relating to psoriasis vulgaris with the use of microsatellite polymorphism markers, which are designed at intervals of about 100 kb, narrowing down candidate regions and then conducting a correlation analysis and a chain analysis with the use of SPNs as markers.

Description

Marker gene for psoriasis vulgaris

The present invention relates to a psoriasis-related gene newly identified using microsatellite genetic polymorphism markers distributed genome-wide and a test method using the gene.

Psoriasis vulgaris is a skin disease characterized by cutaneous keratinization and inflammation associated with abnormal growth of epidermal keratinocytes. After onset, psoriasis vulgaris is an intractable disease with unknown causes that continues to relieve symptoms and relapse. The estimated number of psoriasis patients in Japan is said to be 50,000 to 100,000, and the number of cases registered with the Japanese Psoriasis Society in 1997 is 21,726. The age of onset of psoriasis has an almost symmetrical age distribution peaking at 35 years old, but when limited to women, it shows a bimodality peaking at 10 years old and 50 years old. There is a report. The gender difference is 2: 1 (male: female), which is common among men, but it is not constant in reports from other countries, and it is not necessarily common among men. The prevalence of psoriasis is estimated to be 0.05-0.1%. The most prevalent racial groups and regions are 5-10% for Russian and Norwegian Caucasians and 2.0-3.0% for American, British and Dutch Caucasians. On the other hand, it is reported that North American Indian, Latin American Indian, Mongoloid, and West Africans have the lowest incidence of 0 to 0.3%. Thus, the white population is known to have a higher prevalence than the Japanese population.

Psoriasis is a chronic inflammatory keratosis disease of the skin and is classified as inflammatory keratosis. The inflammatory keratosis is characterized by thickening and keratinization of the epidermis and inflammatory cell infiltration. Histopathological findings of psoriasis include abnormal growth of epidermal keratinocytes, infiltration of inflammatory cells, and vascular proliferation. A reddish phase occurs at the site of frequent occurrence, and scales adhere to the surface. Even in areas where no eruption occurs, eruption is induced by minute trauma (Kobner phenomenon). This Kobner phenomenon is considered to be a change caused by the tissue repair reaction of epidermal cells and dermal tissue destroyed by trauma. In addition, the red phase expands and occurs frequently and merges, chronically. Occasionally with arthritis, transition to pustular psoriasis or psoriatic erythroderma. Subjective symptoms may include rash, itching, painful joint symptoms, and fever.

According to the research on the pathogenesis of psoriasis conducted to date, the epidermis and dermis tissues of the psoriatic lesions include abnormally proliferating keratinocytes, cytokine-producing T cells, activated antigen-presenting cells, Alternatively, it has been shown that there is an increase in immunocompetent cells such as multinucleated leukocytes. In addition, the state of self-proliferative immune response is persistent in the skin eruption, and the balance of various cytokines is lost from the normal state, causing differentiation of keratinocytes and abnormal proliferation, causing inflammatory reaction and immunocompetent cells. Inducing a trigger is considered the cause of the vicious cycle of the condition. Whether cyclosporine, an immunosuppressant, is effective in treating psoriasis, reflecting this pathogenesis, in recent years there have been many reports complaining of its side effects, and no definitive drug has been reached.

In this way, the existence of an immune induction mechanism is becoming clear, but antigens that induce psoriasis-specific immune responses have not been identified, and there is still no comprehensive theory that can explain the pathologically immunologically. I can't see it. In addition, no complete animal model exists. Therefore, it is not clear which factors play an essential role in the onset of psoriasis, the underlying cause and its treatment are unclear, and symptomatic treatment is now the focus.

On the other hand, it is clear that psoriasis has a genetic factor. In 1931 Hoede reported for the first time using genetic statistics that psoriasis had incomplete penetrance and dominant inheritance. About 30 years later, in 1963, a group analyzed in North Carolina, USA, using a very large family, concluded that psoriasis was transmitted through many generations and was inherited dominantly with a penetration rate of about 60%. ing. A very large epidemiological study of psoriasis in Faroe islands, Denmark, reported that year, surveyed 1915 families and 2341 families, which was 1/3 of the population. Among them, 314 psoriasis patients were included (morbidity rate 2.8%), and the onset frequency in the family was as high as 91%. These results ensured the existence of genetic determinants in psoriasis. In Denmark in 1978, a study was conducted on twins with psoriasis. As a result, 72% of monozygotic twins agreed with the onset of psoriasis, but only 15% of dizygotic twins, considering the prevalence rate in Denmark (2 to 3%). Heritability was equivalent to 90-100%. A similar study was conducted in the United States in 1974, and the conclusion was almost the same as the result in Denmark (Non-Patent Document 1). All of these results suggest the involvement of genetic factors. Furthermore, monozygotic twins with similar onset tend to have similar onset ages and distribution of skin eruptions, which is different from that of dizygotic twins (Non-patent Document 2). These are thought to be due to genetic factors affecting certain pathologies in psoriasis, and psoriasis develops in a mixture of multiple genetic factors and some environmental factors Suggests that.

Analysis of the causative gene of psoriasis vulgaris that is thought to include such complex genetic factors has been advanced, and several susceptibility gene candidates have been found. The present inventors elucidated polymorphisms throughout these genes by analyzing in detail the sequences of the MHC S gene, SEEK1 gene, and HCR gene of Japanese psoriasis patients and healthy individuals, and As a result of analyzing the relationship, it was found that some of the analyzed polymorphisms were significantly correlated with psoriasis in the Japanese population (Patent Document 1).
As a method for identifying a new disease-related gene or the like, a technique for examining the correlation between a base and a disease showing single nucleotide polymorphisms (SNPs) present in a human genomic DNA sequence has attracted attention. However, since SNPs are single base substitutions on the genome, the number of alleles is generally only two, and only some SNPs present within about 5 kb from the disease-related gene to be mapped are correlated. In order to perform genome mapping using as a genetic polymorphism marker, it is necessary to set and analyze a huge number of SNPs as markers. Therefore, the present situation is that it is applied only to a narrow region narrowed down to some extent. On the other hand, microsatellite genetic polymorphism markers are characterized by a large number of alleles and a correlation even if they are located at some distance from the gene to be mapped. As in the case of SNPs, the analysis is difficult in terms of time and labor, and if the set number is too small, the marker interval becomes too large and there is a risk of overlooking the disease-related gene.

The present inventors have developed a gene mapping method using genetic polymorphism markers of microsatellite set at intervals of about 50 kb to 150 kb on average, and have disease-related genes or genetic factors by using the method. It was found that the presence region of a human phenotype-related gene can be identified with high efficiency and low cost (Patent Document 2). The present inventors succeeded in identifying a new gene that is considered to be a causative gene of rheumatoid arthritis by genome-wide mapping using this method (Patent Document 3).

Brandrup F, Hauge M, Henningsen J and Eriksen B .: Psoriasis in an unselected series of twins. Arch Dermatol. 1978; 114: 874-878 Farber EM, Nall ML, Watson W .: Natural history of psoriasis in 61 twin pairs. Arch Dermatol. 1974 Feb; 109 (2): 207-11 International Publication WO01 / 042458 Pamphlet International Publication WO01 / 077942 Pamphlet JP 2005-278479 A

The purpose of this study is to identify genetic factors of psoriasis vulgaris, that is, to identify susceptibility genes using a genome-wide mapping method using microsatellite developed by the present inventors. The present invention provides a test method or test kit for susceptibility to psoriasis vulgaris using a susceptibility gene as a marker, and a screening method for a substance useful for the prevention and / or treatment of psoriasis vulgaris.

In the present invention, by using a genome-wide gene mapping method using microsatellite (hereinafter referred to as “MS”), a new susceptibility gene that has not been previously known to be associated with psoriasis vulgaris was identified. .
The common psoriasis susceptibility genes newly identified by the present invention are the OR2J3 gene at locus 6p22.1 of chromosome 6 and the gene IGSF4D at locus 3p12.3 of chromosome 3. The present inventors conducted a correlation analysis between the polymorphisms of these newly identified marker genes and psoriasis vulgaris and found a statistically significant correlation for the first time.

Therefore, as a first aspect, the present invention comprises a continuous DNA partial sequence containing at least one base showing a polymorphism present in the OR2J3 gene or IGSF4D gene of a human genomic DNA sequence, or a complementary strand of the DNA partial sequence Provide a marker gene for testing psoriasis vulgaris.
As a second aspect, the present invention provides a test method and test kit for psoriasis vulgaris using the marker gene.

It is a schematic diagram which shows the genome-wide gene mapping method used by this invention. It is a figure which shows the chromosome position of the positive marker in the tertiary screening using a microsatellite. It is a figure which shows LD map by the SNP marker of 6p22.1. It is a figure which shows the result of Individual typing in 6p22.1. It is a figure which shows the analysis result by the microsatellite and SNPs marker in 6p22.1. It is a figure which shows LD map by the SNP marker of 3p12.31.

The gene mapping method used in the present invention is the method described in Patent Documents 2 and 3. Specifically, as schematically shown in FIG. 1, Pooled using 27,158 microsatellite (MS) markers arranged on a genome-wide basis with 125 samples each of patients and healthy subjects as one analysis set. Perform 3-step screening by DNA typing. In the primary (1st) screening, genetic correlation analysis was performed for all superset markers, and only for markers (positive markers) whose allele frequency difference was statistically significant (P <0.05), secondary (2nd) Perform screening. Furthermore, a tertiary (3rd) screening is performed for a marker that is positive in the secondary screening. Individual positive typing is performed on 375 specimens of all patients and healthy individuals used in this three-stage screening, with respect to the positive markers in this tertiary screening. This is because the allele frequency in Pooled DNA typing is only an estimate, and this allele frequency is determined by this experiment. A specific analysis method at each stage will be described in detail in Examples described later.

Correlation with psoriasis vulgaris confirmed by the above method is the OR2J3 gene at locus 6p22.1 of chromosome 6 and IGSF4D at locus 3p12.3 of chromosome 3. Furthermore, the correlation between the OR2J3 gene and the HLA-C gene, which has been conventionally known to be associated with psoriasis vulgaris, has been clarified for the first time.
Further, after screening a DNA sample from a subject using the marker gene of the present invention as a probe and determining the nucleotide sequence of the obtained subject DNA, the sequence is compared with the sequence of a healthy person, thereby also preventing psoriasis vulgaris The presence or absence of a genetic predisposition can be examined.

The probe used here may be the marker gene itself of the present invention, but is a continuous DNA sequence containing a base represented by a single nucleotide polymorphism present in the marker gene or a complementary strand thereof, or a sequence hybridizing to them. Also good. A probe having a length of preferably 15 to 100 bases, more preferably 15 to 25 bases, and further preferably 18 to 22 bases can be used.

On the other hand, based on the OR2J3 gene or IGSF4D gene newly found in the present invention, by determining the full-length base sequence of the psoriasis vulgaris susceptibility gene, the translation region encoded by them can be determined. The amino acid sequence of the encoded protein can be specified. Since proteins with these amino acid sequences are likely to be involved in the pathogenesis or pathogenesis of psoriasis vulgaris, prevent or treat psoriasis vulgaris by promoting or inhibiting the function of these proteins be able to.
Therefore, the present invention also relates to a screening method using the protein, and the screening method can identify a substance that promotes or inhibits the function of the protein, that is, an agonist or an antagonist. The term “antagonist” as used herein includes not only small chemical molecules but also biologically relevant substances such as antibodies or fragments thereof, and antisense oligonucleotides. These agonists or antagonists are effective as diagnostic, prophylactic and / or therapeutic agents for psoriasis vulgaris.

For the above protein, a sugar is produced in the transformed cell by preparing a vector containing a DNA sequence containing at least the translation region of the marker gene identified in the present invention, and transforming an appropriate host cell with the vector. It is also possible to make it.

Hereinafter, specific examples will be shown, and the present invention will be described in detail based on the results.
1. experimental method:
(1) DNA extraction / quantification / mixing All DNA used for genotyping was prepared by the following method. That is, genomic DNA was extracted and purified from the collected blood using the QIAamp DNA Blood Maxi Kit (QIAGEN). Elution of genomic DNA from the column was performed using TE (10 mM Tris-HCl, 0.1 mM EDTA) having an EDTA concentration of one-tenth the usual amount in consideration of the influence on the amplification efficiency of PCR. After extraction, the purity was confirmed by agarose gel electrophoresis and no degradation of DNA, and the 260/280 ratio of the absorbance measurement result.

The amount of DNA and the preparation of a mixed DNA (pooled DNA) solution were performed according to the following procedure. As a method for quantifying DNA, a method using a fluorescent dye PicoGreen reagent (Molecular Probes) that specifically stains double-stranded DNA and a fluorescent plate reader was used. A λ phage DNA attached to the PicoGreen reagent was used as a standard sample at the time of concentration measurement, and a 5-point dilution series (10, 30, 100, 300, 1000 μpg / mL) was used. Each individual's genomic DNA was diluted 400 times and quantified three times. Based on the results of the three quantifications, a total of three combinations of two doses were made, and the average value, S.D. and C.V. A combination having a C.V. of 5% or less and the lowest value was selected, and the average value was adopted as the final DNA concentration. When C.V. exceeded 5% in any of the three combinations, DNA quantification was repeated until a combination of 5% or less was obtained.

Based on the DNA concentration determined through such quantitative operation, 125 individual DNAs were mixed in a certain amount of DNA, TE (10 mM Tris-HCl, 0.1 mM mM EDTA) was added, and finally 8 ng / μL. pooled DNA. In order to enable primary screening and secondary screening using pooled DNA, two sets of such pooled DNA consisting of 125 individuals were prepared for each patient and healthy subject. The sex ratio of 125 individuals in each set is the same between the patient's pooled DNA and the healthy person's pooled DNA, and the age frequency distribution is as close as possible between the two in the 10-year class. So elected.

(2) Pooled DNA genotyping
The primer set for each marker used was a forward primer with a 5 ′ end fluorescently labeled with 6-FAM or HEX, and an unlabeled reverse primer. All primers were manufactured by Proligo.
The reaction solution was prepared as follows. That is, in a total volume of 20 μL, 24 ng of pooled DNA (3 μL of 8 ng / μL), 2 μL of 10 × buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl 2), 2 pmol A forward primer and a reverse primer were prepared to contain 0.5 U of AmpliTaq DNA polymerase (Applied Biosystems). The PCR cycle was 96 ° C for 5 minutes, 56 ° C for 1 minute, 72 ° C for 1 minute, 1 cycle, 96 ° C for 45 seconds, 57 ° C for 45 seconds, and 72 ° C for 45 seconds for 40 cycles.

Depending on the amplification efficiency, PCR products are diluted 20-fold or 40-fold with ultrapure water, dried using a vacuum pump or evaporator, then formamide (Applied Biosystems) and DNA size marker GS500 ROX (Applied Bio). System)). After heat treatment at 96 ° C. for 5 minutes, electrophoresis was performed with a DNA analyzer ABI-3700, and the size determination (sizing) of each fluorescent signal peak derived from the PCR product was performed with GeneScan® analysis software (Applied Biosystems). After sizing, the size and height data of each peak was extracted using PickPeak software.

(3) SNP genotyping
SNP information in the susceptibility gene candidate region found by analysis with a microsatellite marker was selected and extracted from NCBI (http://www.ncbi.nlm.nih.gov/). A total of 940 specimens including patients and healthy individuals used in the experiment with the polymorphic microsatellite marker were used, and the genotype of the SNP was determined by the TaqMan method and the direct sequencing method. The TaqMan method was operated with a standard protocol using an ABI7900HT (Applied Biosystems) system. The direct sequencing method used an ABI3700 (Applied Biosystems) sequencer, which also used a standard protocol.

(4) Statistical calculation To compare the estimated allele frequencies obtained from pooled DNA consisting of patient samples and pooled DNA consisting of healthy subjects for each marker, χ ² test and Fisher's direct probability test Statistical processing was performed according to the following procedure.
For each certain gene locus (marker), the sum of the heights of the fluorescent signal peaks derived from the detected PCR products was determined. Using this as the denominator, the ratio of the height of each allele peak was calculated and used as the gene frequency within the population for each allele. Statistical processing was performed by calculating the estimated number of alleles in the population by multiplying the total number of alleles of the samples contained in the pooled DNA, and using this allele number. Specifically, 2 × 2 and 2 × m contingency tables were created based on the number of alleles obtained, and χ ² test and Fisher's direct probability test were performed to detect markers showing a significant correlation.

These methods were also applied to individual typing of microsatellite markers and SNP genotyping. Furthermore, LD (Linkage Disequilibrium) blocks were constructed from SNP markers using D'value confidence intervals. For this purpose, the estimation of haplotypes in each LD block was obtained using EM and Clark algorithms. That is, based on the estimated haplotype frequency, a 95% confidence interval was calculated from each haplotype frequency distribution given by performing 2,000 resamplings by the bootstrap method.
As additional samples other than Pooled DNA typing and Individual typing, 186 samples were used for each patient and healthy subject. Haploview (JC Barrett, B. Fry, J. Maller, MJ Daly: Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics, 2005, Jan., 15: 21 (2): 263-5) was used.

2. Experimental results and discussion:
The positive markers detected by the 1st screening were 2,065 markers among all polymorphic microsatellite markers. In the 2nd screening, 495 markers out of these 2,065 markers were positive. Furthermore, 158 markers out of these 495 markers were positive in the 3rd screening (Table 1). The number of these positive markers is slightly higher than that observed in rheumatoid arthritis, but remains almost unchanged. Therefore, although it was more than the number expected statistically, it was considered to be an experimental false positive that still depends on the Pooled DNA typing method.

The position of this positive 158 marker on the chromosome is shown in FIG. In FIG. 2, P value is shown on a logarithmic scale, and positive markers are indicated by circles. The bar above the chromosomal band indicates the psoriasis susceptibility region suggested by the previously reported linkage analysis. As described above, psoriasis has a related gene in the HLA region 6p21.3. In this analysis, a positive marker was also observed in this region, and it was proved again that the present research method functions correctly.

Subsequently, the estimated allele frequency was determined by individual-typing and experimental false positives were excluded. As a result, it was narrowed down to 42 positive markers in Pooled DNA typing. Furthermore, among these, 17 markers (see Table 2) whose P values obtained from the contingency table of 2 × 2 and 2 × m 共 に are both 0.05 or less are judged to be particularly strongly associated with the disease, It was set as the analysis object area after that.

Subsequently, SNP-typing was performed to narrow down these regions in detail and finally identify susceptibility genes. Of the 17 regions, 15 regions excluding D12S1638 and D2S0276i were selected from the vicinity of the markers so that each SNP marker could be placed on a few kilobases.

6p22.1
The D6S0091i marker present in this region shows the lowest P value in the genome-wide marker group (P = 0.000004), which is more relevant than D6S0076i in the HLA class I region, which is a known sensitive region Met. In the 300 kb region centered on this D6S0091i marker, 74 SNPs with a Minor Allele frequency (MAF) of 0.05 or more were finally selected (TaqMan: 63, Sequencing: 9) and then pooled DNA typing. Genotyping was performed on the used patients and 750 healthy subjects and 372 additional samples. However, almost no SNPs showing a significant allyl frequency difference were detected, and even the lowest P value SNP (rs3117329) was 0.038 (allele frequency). The results of LD analysis using the Haploview program based on these data are shown in FIG.

As a result of defining LD block in Four Gamete Rule, 5 LD blocks were estimated. A significant difference test was performed on the frequency of haplotypes in patients and healthy individuals in each block, but only one haplotype in block 1 with P 示 す value of 0.035 was detected, and no haplotype that was considered to be related to psoriasis was detected. It was.

On the other hand, the marker of D6S0091i was present at 995 bp 5 'upstream of the first codon of the OR2J3 gene, and it was considered that the allele of this microsatellite itself may regulate psoriasis sensitivity itself. Therefore, SNPs between this marker and the OR2J3 gene up to 3'UTR (about 2 kb) and polymorphisms such as Insertion and Deletion are all detected by sequencing, and the microsatellite sequence of about 130 kb is centered on this marker. Attempts to exclusively identify the psoriasis susceptibility locus as well as the allele by detecting all polymorphisms.

As a result of sequencing, one 3-base deletion and 24 SNPs were detected. Of these, 12 were in the coding region of the OR2J3 gene and 8 were amino acid substitutions. Nine Minor Allele frequency was 0.05 or more. When statistics were calculated for these SNPs, the P value (0.015) in the C dominant model of SNP (T / C) present at 745 bp upstream 5 ′ of the first codon was the lowest, but D6S0091i Since it is unlikely that the association with psoriasis is stronger than the marker, it was considered that there was no sensitive allele on the OR2J3 gene.

On the other hand, when 23 microsatellite sequences were found from the genome sequence and a polymorphism search was performed, polymorphism was shown in 5 microsatellite. With these five microsatellite markers and D6S0091i markers, and two genome-wide markers (D6S258, D6S1683) set outside these, individual-typing was performed on all samples of patients and healthy subjects. These results are shown in Table 3 and FIG.

The P value of the marker of the genome wide marker group D6S0091i was the lowest (0.0000000065), and the Odds Ratio at 469 considered to be a risk allele was 2.16. The P value of the MS15 marker that found a new polymorphism was the next lowest 0.000.00. On the other hand, none of the other microsatellite markers showed a significant difference between patients and healthy subjects. That is, since these two markers are located at a very short distance upstream and downstream across the OR2J3 gene, the OR2J3 gene was determined as a psoriasis susceptibility gene present in 6p22.1. Furthermore, since the allele frequency of 350, which is considered to be a risk allele of MS15, is extremely low, the sensitive allele was considered to be 469 of the marker for D6S0091i.

In an experiment on the above Japanese population, it was revealed that allele 469 of microsatellite D6S0091i located in the sensitive region 6p22.1 correlates strongly with psoriasis vulgaris.
Since correlation analysis is a multiple test, correction is generally required for statistical tests performed. However, when a very large number of genetic markers are used, the number of tests becomes very large, so that correction is practically impossible. On the other hand, it is possible to improve the accuracy of data by performing a supplementary test on the correlation with the patient obtained in one ethnic group in different ethnic groups. Therefore, we examined below whether or not D6S0091i allele 469, which was found among psoriasis patients and healthy individuals in the Japanese population, could be tested by other ethnic groups.

Using the DNA prepared from the peripheral blood of 102 patients with psoriasis in the Mongolian population and 168 healthy individuals, the microsatellite marker D6S0091i was typed by a standard method to determine the allele of each individual. The results are shown in the table below.

Even in the Mongolian population, this D6S0091i had a polymorphism, and the allele frequency distribution approximated that of the Japanese population. Furthermore, in the Mongolian population, allele 469 was correlated with psoriasis as in the Japanese population (see Table 4). When Fisher's Exact test was performed from a 2 × 2 contingency table, the p value was 0.002 and the Odds Ratio was 2.40 (p value = 0.0000000065, Odds Ratio = 2.16 in the Japanese population).
From the above results, the correlation observed in the Japanese population was also observed in the Mongolian population. In other words, the follow-up test in other ethnic groups was successful, and it was strongly supported that D6S0091i allele 469 correlates with psoriasis.

Furthermore, the frequency of the allele in the white population (healthy subjects) collected in Australia was 13.9% (190 people), slightly higher than that of the Japanese, and the distribution of the nine allele frequencies was similar.

As described above, regarding 6p22.1, we successfully identified mutations other than SNP exclusively as sensitive alleles by intensively covering SNPs and microsatellite markers. In addition, considering the process leading to identification of this sensitive allele, the OR2J3 gene cannot be found as a sensitive gene in genome-wide mapping as long as the SNP marker is used for the initial screening, and can be obtained for the first time by the present invention. This is an advantageous effect.

The OR2J3 gene belongs to the olfactory receptor (OR) gene family. It is estimated that there are about 900 OR genes in the human genome, of which 53% to 63% are pseudogenes and are present on all chromosomes other than No. 20 and Y. In addition, about 80% of OR genes form 6 or more gene clusters, and it is already known that this OR gene cluster exists in 6p22.1, and OR2J3 gene is included in this. . The OR gene has no intron and the coding is about 1 kb, whereas the OR2J3 gene is thought to have a coding region of 936 bp and define 311 amino acids. Since the OR gene is a G protein-coupled receptor having seven transmembrane helices, it is speculated that the OR2J3 gene also functions as some kind of receptor.

As described above, since the D6S0091i marker having a psoriasis-susceptible allele is located at 995 bp 5 'upstream of the first codon of the OR2J3 gene, it is very likely to be in the transcriptional regulatory region of this gene. Therefore, it is considered that elucidation of the mechanism of psoriasis vulgaris is further promoted by investigating the expression level by introducing a gene region containing a sensitive allele into cells and analyzing the lesion in a patient.

6p21.3
Psoriasis has been reported across races to correlate with HLA-Cw ^* 0602 in the HLA class I region, and many other results suggest that this region is almost certainly a psoriasis-sensitive region. Yes. On the other hand, no conclusion has been reached as to whether the susceptibility gene is the HLA-C gene itself, another gene in the vicinity thereof, or whether there are multiple genes.

Of the 17 positive markers narrowed down by individual typing shown in Table 2 above, the D6S0076i marker was located in the vicinity of the CDSN gene considered as one of the psoriasis susceptibility genes. Therefore, in order to narrow down the sensitivity region in more detail, an attempt was made to analyze by arranging microsatellite markers and SNPs markers at high density. That is, nine microsatellite markers (1 / 6.1.1 kb) including the D6S0076i marker in this region of about 500 kb and 98 SNPs markers (1 / 4.8 kb: MAF ≧ 0.05) were used. Further, additional microsatellite markers were carefully selected by referring to public reports and SNPs markers by referring to public databases. All samples of patients and healthy subjects were subjected to genotyping by individual typing for microsatellite markers and TaqMan method for all SNP markers.
As a result of individual typing with microsatellite markers, strong correlations that retain statistical significance were detected in 7 markers (400 kb) from C4_2_7 to C1_2_5 markers even after Bonferroni's correction was performed (Table 5 below and FIG. 5 ( (See ■).

We performed a correlation analysis using a population of psoriasis patients with microsatellite markers placed only in the HLA region, and found significant frequency differences between C4_2_12 and C1_2_6 markers (located between C1_3_2 and C1_4_4) (Oka A, Tamiya G, Tomizawa M, Ota M, Katsuyama Y, Makino S, Shiina T, Yoshitome M, Iizuka M, Sasao Y, Iwashita K, Kawakubo Y, Sugai J, Ozawa A , Ohkido M, Kimura M, Bahram S, Inoko H .: Association analysis using refined microsatellite markers localizes a susceptibility locus for psoriasis vulgaris within a 111 kb segment telomeric to the HLA-C gene.Hum Mol Genet. 1999 Nov; 8 (12 ): 2165-70). Although the results of this study succeeded in reexamining the previously indicated region, a marker that was also strongly correlated to the centromere side was found. This is because the C1_2_5 and C1_4_3 markers have been observed to have a significant correlation before the statistic correction even in the previous results, and the number of samples used has increased significantly (the total number of patients and healthy subjects has increased from 208 to 1,122). This is thought to be due to improved statistical power.
Next, in order to obtain the basic findings of the psoriasis study for all the specimens used in this example, the allele frequency of the HLA-C locus was investigated (Table 6).

As a result, although the allele frequency was low, the strongest correlation was observed in Cw * 0602 (P = 0.00000000019). On the other hand, the frequency of this allele is as high as 10 to 20% in the Caucasian group and less than 1% in the Japanese group. Reflecting this fact, there is a report that the frequency in the population of Cw * 0602 and the prevalence of each country have a positive correlation. In addition, the allele frequency in the patient group increased, and even if Bonferroni's correction is performed, the only allele that retains statistical significance is Cw * 0102, in addition to Cw * 0602, and even the most frequent allele in the population. there were. Cw * 0102, which showed this significant correlation, is also the allele that was found to be associated with psoriasis for the first time in this study.

Estimate LD block (Four Gamete Rule) from the data obtained from SNP genotyping, and calculate each statistic in Table 7. Also, this data and the result of individual typing by the above-mentioned microsatellite marker, and gene map The combination is shown in FIG. In FIG. 5, the upper part of the gene map is described in the direction of centromere on the left side and the direction of telomere on the right side, and the lower part shows P in the frequency difference test between healthy subjects calculated from the allele frequencies of each SNP and each microsatellite marker. Value was plotted. In addition, the estimated LD block position is shown.

As a result of evaluating the frequency difference from the allele data of each SNP marker by Permutation test (calculation of corrected P value) implemented in Haploview, 22 out of 98 SNPs having a P value of 0.05 or less were present. (Figure 5: Indicated by ◆). In addition, regarding the estimated frequency difference test in the LD block, 11 Haplotypes having a P value of 0.05 or less were found in Permutation test (Fig. 5: B01, B02, B04, B05, B06, B08, B11, B13, B14, B15, B16).

Although the P value of Haplotype · B02 and the SNPs marker contained therein rs7381905 were extremely low, each 10 ^-15 or less, no expressed gene is known in this Haplotype. However, this region overlapped with the 60 kb risk haplotype 1 (RH1) that does not contain the HLA-C locus reported by Nair et al. On the other hand, Hells et al., Whose results are in conflict with this report, have identified the region from the centromere side of the HLA-C locus to the HLA-C locus as a sensitive region. However, since it was only set, detailed evaluation combined with the former data could not be performed. However, analysis using a Japanese population suggested for the first time that a sensitive region may exist in the very close region, including the HLA-C locus itself.

LD blocks B04 to B06 contain POU5F1 and TCF19 genes of transcription factors, and SPR1, SEEK1, and CDSN genes that are expressed specifically in epidermal tissues.
In addition, LD block B13 to B16 have a total of 5 known genes: DPCR1 gene: diffuse panbronchiolitis candidate gene, VARS2L gene: tRNA synthetase, GTF2H4 gene: transcription factor IIH, DDR1 gene: tyrosine kinase receptor Yes. In addition, there is no previous report that genetic markers in this region correlate with psoriasis, and it was first discovered in this study and is very interesting.

In this experiment, many markers and Haplotypes showing extremely significant correlation are found in the HLA class I region, and it appears as if there are multiple sensitive regions. Helms et al. Reported that only the region containing the HLA-C locus was a sensitive region and the others were independent, Nair et al. Claimed not to contain the HLA-C locus, and Orru et al. It concludes that it is around 70kb (Orru S, Giuressi E, Carcassi C, Casula M, Contu L .: Mapping of the major psoriasis-susceptibility locus (PSORS1) in a 70-Kb interval around the corneodesmosin gene A J Hum Genet. 2005 Jan; 76 (1): 164-71). Thus, there is no unified consensus on psoriasis genetic research in the HLA region. About 400 kb defined by the seven microsatellite markers that have been correlated is one linkage disequilibrium region, and it is not clear at this time whether there is one or more susceptibility loci.

1. Interaction between OR2J3 gene and HLA-C gene When tracking susceptibility genes of multifactorial diseases targeted in the present invention by correlation analysis, etc., multiple loci may contribute to the onset simultaneously or in stages. It is thought that it is necessary to evaluate each interaction in consideration of the sex, and several methods have been proposed (Marchini J, Donnelly P, Cardon LR .: Genome-wide strategies for detecting multiple loci that influence complex diseases Nat Genet. 2005 Apr; 37 (4): 413-7).
Therefore, an attempt was made to investigate the interaction between the OR2J3 gene newly found as a susceptibility gene in this study and the HLA-C gene. In this study, we divided into a group with and without an allele at one locus and evaluated the allele frequency at the other locus. In other words, the Confounding factor is analyzed by stratification.

The allelic 469 of the D6S0076i marker present upstream of the OR2J3 gene and Cw * 0102, Cw * 1202, and Cw0602 tending to significantly increase the allele frequency in the patient group were stratified by the presence or absence of each allele, The results of a comparative study of changes in Odds Ratio are reported below.
First, Table 8 shows the analysis results of sensitive allele 469 and Cw * 0102.

The Odds Ratio of the sensitive allele 469 in the non-stratified population (All Population) is 2.160, whereas in the population without Cw * 0102, the Odds Ratio drops to almost 1, but the population with Cw * 0102 Then, Odds Ratio is rising to 2.858. On the other hand, the Odds Ratio of Cw * 0102 in the non-stratified population is 1.384, whereas the Odds Ratio in the population without the sensitive allele 469 decreases by almost 1, but in the population with the sensitive allele 469, The Odds Ratio has risen to 1.959. In other words, the difference in the allele frequency between patients and healthy subjects in the group without any allele disappeared, but the odds ratio was significantly increased in both groups with allele (statistically significant). There is a difference). Therefore, it was suggested that these two alleles may interact and lead to the development of psoriasis.
Next, there was no statistically significant frequency difference (P value after Bonferroni's correction) (Table 5), but the analysis results of Cw * 1202 and sensitive allele 469 tending to increase in the patient group are shown. It is shown in FIG.

There is almost no change in Odds Ratio in the non-stratified population and the population stratified with Cw * 1202, and conversely, there is no significant change in Odds Ratio in the population stratified with sensitive allele 469 (either There was no statistically significant difference), and no interaction between these alleles could be confirmed.

Furthermore, Cw * 0602 (Table 5), which showed a very significant frequency difference, was examined even though its allele frequency was low (Table 9).

There was almost no difference in the Odds ratio when stratified into a group that was not stratified and a group that did not have Cw * 0602. In other combinations, since there were no individuals with Cw * 0602 in the healthy population, Odds Ratio could not be calculated. However, focusing on the allele frequency in the patient population, the allele frequency tended to be higher in the group without any allele than in the non-stratified population. That is, Cw * 0602, which has a relatively high frequency and a very strong correlation in the Caucasian population, etc., may have no interaction with the sensitive allele 469 or may have a negative interaction. .

An analysis by stratification of sensitive alleles 469 other than these three combinations with alleles of each HLA-C locus was also performed, but no allele combination that strongly interacted was found. That is, it was revealed that only Cw * 0102 among the sensitive allele 469 and the HLA-C gene shows the possibility of interaction.

3p12.3
In addition to 750 patients and pooled DNA typing used in pooled DNA typing, 372 additional specimens were subjected to genotyping with the D3S0536i marker, indicating a stronger correlation (P = 0.00040). The Odds Ratio at 251 considered to be risk allyl was 0.60.
37 SNP markers (MAF ≧ 0.05, 1 / 5.5 kb) were arranged over 203.2 kb centering on this marker, and genotyping was performed on all patients and healthy populations. As a result of analysis such as estimation of LD, four LD block (Four Gamete Rule) were estimated (FIG. 6).
Moreover, as a result of examining the difference in the frequency of Haplotype between patients and healthy subjects in the estimated LD block, the most significant correlation was shown in Block 1 (FIG. 6, Table 11).

On the other hand, the most significant correlation was shown in the rs7643091 marker contained in Block 2 in the test of the allele frequency difference with the SNPs marker alone, not the estimated Haplotype, but the P value was 0.003, Permutation test was 0.051 and did not show stronger correlation than Block 1. Therefore, it is considered that this Haplotype itself functions as a disease susceptibility or there may be a SNP that is more strongly correlated within Block 1, that is, a sensitive allele.

There is only one known gene in this region, which is the gene name IGSG4D. The protein encoded by this gene is called SynCAM2. Therefore, the psoriasis susceptibility gene present in this region is considered to be the IGSF4D gene.
This gene is predicted to have a total genome length of 1.11 Mb, 10 exons, 3,948 bp as a cDNA, 1,308 bp as coding, and 435 amino acids as a protein. The 203.2 kb region to be analyzed this time is located in the first intron (843 kb) of this gene. That is, it is very unlikely that a polymorphism involving amino acid mutation or the like is a sensitive allele, and polymorphisms on regulatory elements such as transcription factors and enhancers are presumed to contribute to the onset. On the other hand, the Odds Ratio of the found Block 1 is 0.59, and the D3S0536i marker is 0.60, and the target haplotype or allele was less frequent than the healthy group in the patient group and was negatively correlated. Was probably suppressed against the onset of psoriasis.

SynCAM2 encoded by IGSF4D is classified into the immunoglobulin superfamily, which is a large protein group. Furthermore, this protein is one of four SynCAM protein groups.
The extracellular region of the SynCAM2 protein is defined by two Ig-like domains connected by a short linker region immediately after the signal peptide from the N-terminus. Furthermore, following the signal transmembrane region, the cell substrate tail is formed.

-Other candidate regions Fifteen regions that have been narrowed down by microsatellite markers and have completed experiments and analysis using SNPs markers, of which three were as described above, succeeded in finding a psoriasis susceptibility gene.
For the remaining 12 regions, total 2829 kb, all specimens (patient: 561, healthy subjects) were obtained by TaqMan method using 437 SNP markers that finally had heterozygosity of 0.1 or more and had no problems in experiments and analysis. 561) was genotyping (D17S0178i, D2S0233i, and D6S0802i were only additional populations). As a result, a significant difference test was performed on the allele frequency, the genotype frequency, and the frequency in the dominant model of each of the two alleles between the patient and the healthy subject. A total of 68 markers were observed (Table 12).

As a result of analysis of LD block, the estimated number of LD block was 88 in total, and among them, the number of Haplotype in which a significant difference was found in the Haplotype frequency between patients and healthy subjects was 18 (Table 12). . However, when all these tests were performed by Permutation test, there were no SNPs or Haplotypes that showed significant frequency differences. Therefore, in this analysis, we could not find data that strongly suggests the presence of susceptibility genes in these regions.

However, the number of SNPs and Haplotypes showing significant differences found by the Chi-Square test test is clearly higher than the estimated number of type I errors and may contain true sensitive alleles or Haplotypes. Sex cannot be denied. In particular, in the region of the D4S0178i marker, among the 39 SNPs markers, there is a significant correlation among 4 haplotypes among 25 LDs and 7 LD blocks.

Preparation of antibody It was revealed that allele 469 of microsatellite D6S0091i located in the sensitive region 6p22.1 found by genome-wide correlation analysis strongly correlates with psoriasis vulgaris. Moreover, since this D6S0091i is present at 995 bp 5 ′ upstream of the first codon of the OR2J3 gene, this OR2J3 gene is strongly suspected as a gene that regulates susceptibility to psoriasis. However, although this gene has been named as an olfactory receptor, it has only been predicted from the DNA sequence and has not been analyzed experimentally and its function is unknown.
In order to analyze whether this gene encodes an amino acid, actually functions as a protein, and what physiological activity it can have, it is necessary to prepare a specific antibody of the protein that this gene is thought to produce . If an antibody is obtained, the antibody can be used to contribute to the prevention or treatment of psoriasis vulgaris by promoting or suppressing the function of the protein encoded by the susceptibility gene. Therefore, mice were immunized with the peptide as an antigen to produce an anti-OR2J3 monoclonal antibody.

This OR2J3 gene is one of a large gene family, and the homology of each gene sequence and amino acid sequence is extremely high. Therefore, we carefully selected an amino acid sequence that is specific for this gene, and adopted a strategy of immunizing mice with antigens as peptides to produce monoclonal antibodies.
In preparing a peptide as an antigen, it is important which amino acid sequence is targeted. In order to produce an antibody against the protein derived from the OR2J3 gene, the following four points were observed.
1. Avoid crossover with highly homologous olfactory receptor proteins.
2. Avoid sites with amino acid mutations due to previously discovered SNP polymorphisms.
3. Avoid sites that may undergo glycosylation.
4). Select a site that is predicted to be highly antigenic.

Only one part satisfies the above four conditions. The OR2J3 protein is presumed to be composed of 311 amino acids, and among them, 11 amino acids from the 263th to the 273rd were selected as peptides having the antigen. The amino acid sequence of this peptide is “LQPPSGNSQDQ”. However, since there is an OR2J1 gene with extremely high homology to this amino acid sequence, a method of selecting a hybridoma that reacts with “LQPPSGNSQDQ” and does not react with “LQPPS E NSQDQ” when screening by ELISA at the time of monoclonal antibody production. It was adopted.

Since peptides are generally less antigenic than other immunogens, it is common practice to immunize by coupling a carrier protein to the peptide. Therefore, a method was employed in which a peptide was synthesized with a “C” residue added to the N-terminus of the peptide and KLH (keyhole limpet hemacyanin) was coupled. That is, the amino acid sequences of peptides synthesized for producing monoclonal antibodies are as follows.
・ Immunogen “CLQPPSGNSQDQ” (SEQ ID NO: 1)
・ "CLQPPS E NSQDQ" for screening (SEQ ID NO: 2)
For final screening, peptides without addition of the aforementioned “C” residue (SEQ ID NOs: 3 and 4, respectively) were also used.

KLH was coupled to “CLQPPSGNSQDQ”, immunized to a C3H background mouse, fused with mouse myeloma cells by a conventional method, and cultured in a selective medium by a limiting dilution method. Seven clones of hybridomas that reacted with the peptide of the immunogen by primary screening by ELISA but did not react for screening were selected and further cultured by limiting dilution.
Among the 7 clones, 2 clones stopped growing in the middle of the culture, and 5 clones remained. A secondary screening by ELISA was carried out. Of these 5 clones, the antibody activity in the culture supernatants of 4 clones was low and judged to be unsuitable for the subsequent analysis. The remaining 1 clone was identified as an anti-OR2J3 monoclonal antibody. It was decided to adopt as a production hybridoma (clone name: LK16-3). The antibody produced by this clone was found to be IgM isotype by ELISA assay. The results of the assay by ELISA using each peptide as an antigen are shown in Table 13 as OD values.

As a result, since the peptide derived from the OR2J3 gene for screening and this LK16-3 hardly reacted, it was confirmed that an anti-OR2J3 monoclonal antibody having extremely high specificity was produced.

Claims (13)

A marker gene for examination of psoriasis vulgaris comprising a continuous DNA partial sequence containing at least one base showing a polymorphism present in the OR2J3 gene or IGSF4D gene of a human genome sequence, or a complementary strand of the DNA partial sequence. The marker gene according to claim 1, which has a length of 50 to 1500 bp. The marker gene according to claim 2, which has a length of 100 to 1000 bp. A DNA partial sequence corresponding to the marker gene according to any one of claims 1 to 3 is collected from a subject, the base sequence of the DNA partial sequence is determined, and the base sequence obtained from a healthy subject A test method for psoriasis vulgaris, comprising comparing to A test kit for psoriasis vulgaris comprising the marker gene according to any one of claims 1 to 3, or a primer thereof. A vector comprising the DNA sequence of the marker gene according to any one of claims 1 to 3. A host cell transformed with the vector of claim 6. A polypeptide encoded by the marker gene according to any one of claims 1 to 3. A method for producing the polypeptide according to claim 8, comprising incubating the host cell according to claim 7 under conditions suitable for expression. The screening method of the chemical | medical agent which diagnoses, treats and / or treats psoriasis vulgaris using the polypeptide of Claim 8. An agonist and / or antagonist obtained by the screening method according to claim 10. A diagnostic, prophylactic and / or therapeutic agent for psoriasis vulgaris comprising the agonist and / or antagonist according to claim 11. The diagnostic, prophylactic and / or therapeutic agent according to claim 12, wherein the agonist and / or antagonist is an antibody.

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