Method for detecting the severity of atopic dermatitis

Abstract

To provide a marker for detecting the severity of atopic dermatitis and a method for detecting the severity of atopic dermatitis using the marker.SOLUTION: A method for detecting the severity of atopic dermatitis in a subject includes a step for measuring, in a biological sample collected from the subject, the expression level of at least one gene, or the expression product thereof, selected from the group consisting of nine genes which are: CAPN1, NCOR2, PPP1R9B, PLP2, GRN, PPP1R12C, LOC146880, SLC31A1 and SYVN1.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for detecting the severity of atopic dermatitis using a marker for the severity of atopic dermatitis. [Background technology]

[0002] Atopic dermatitis (hereinafter also referred to as "AD") is an eczematous skin disease that primarily occurs in people with a predisposition to atopy. Typical symptoms of atopic dermatitis include chronic and recurrent itching, rash, erythema, etc., occurring bilaterally and contralaterally, as well as hypokeratosis, impaired barrier function, and dry skin. Most cases of atopic dermatitis occur in infants and young children, and tend to improve with age. However, in recent years, adult-onset and intractable atopic dermatitis have also been increasing.

[0003] The onset and severity of atopic dermatitis can be evaluated to some extent by visual observation of the skin or image analysis of the skin surface. Recently, tests using blood TARC (thymus and activation-regulated chemokine) levels and SCCA2 (squamous cell carcinoma antigen 2) levels as indicators have been used to objectively assess the severity of atopic dermatitis. These indicators are used to sensitively reflect the progression of atopic dermatitis, and are used not only to evaluate the onset and severity of atopic dermatitis, but also to educate patients and determine treatment strategies (see Non-Patent Documents 1, 2, and 3). However, testing methods that use blood TARC and SCCA2 levels as indicators are invasive because they require blood sampling, and they also have issues such as the time it takes to obtain results.

[0004] Meanwhile, technologies have been developed to investigate the current and future physiological state of the human body by analyzing nucleic acids such as DNA and RNA in biological samples. Nucleic acid analysis has the advantages of comprehensive analytical methods, which enable the acquisition of a wealth of information in a single analysis, and of the ease of functionally linking analytical results based on numerous research reports on single-nucleotide polymorphisms and RNA function. While biological nucleic acids can be extracted from body fluids such as blood, secretions, and tissues, it has recently been reported that RNA contained in skin surface lipids (SSL) can be used as a biological sample for biological analysis, and that marker genes for the epidermis, sweat glands, hair follicles, and sebaceous glands can be detected from SSL (Patent Document 1). Furthermore, it has been reported that marker genes for atopic dermatitis can be detected from SSL (Patent Document 2). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2018 / 008319 [Patent Document 2] Japanese Patent Application Publication No. 2020-74769 [Non-patent literature]

[0006] [Non-Patent Document 1] Sugawara et al., Allergy (2002) 57:180-181: [Non-patent document 2] Ohta et al., Ann Clin Biochem.(2012) 49:277-84 [Non-patent document 3] Kato et al., Journal of the Japanese Dermatological Association (2018) 128: 2431-2502 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention relates to providing a marker for detecting the severity of atopic dermatitis and a method for detecting the severity of atopic dermatitis using the marker. [Means for solving the problem]

[0008] The inventors collected SSLs from patients with atopic dermatitis of different severity and healthy individuals, and comprehensively analyzed the expression state of RNA contained in the SSLs as sequence information. As a result, they found that the expression levels of specific genes differed significantly between patients with different severity and between healthy individuals, and that this could be used as an indicator to detect the severity of atopic dermatitis.

[0009] That is, the present invention relates to the following 1) to 3). 1) A method for detecting the severity of atopic dermatitis in a subject, comprising a step of measuring the expression level of at least one gene or its expression product selected from a group of nine genes consisting of CAPN1, NCOR2, PPP1R9B, PLP2, GRN, PPP1R12C, LOC146880, SLC31A1 and SYVN1 in a biological sample collected from the subject. 2) A test kit for detecting the severity of atopic dermatitis used in the method of 1), which contains an oligonucleotide that specifically hybridizes with the gene or an antibody that recognizes the expression product of the gene. 3) A marker for detecting the severity of atopic dermatitis, consisting of at least one gene or its expression product selected from a group of nine genes consisting of CAPN1, NCOR2, PPP1R9B, PLP2, GRN, PPP1R12C, LOC146880, SLC31A1 and SYVN1. [Effects of the Invention]

[0010] According to the present invention, it is possible to easily and non-invasively detect the severity of atopic dermatitis early, accurately and objectively. DETAILED DESCRIPTION OF THE INVENTION

[0011] All patents, non-patent documents, and other publications cited herein are hereby incorporated by reference in their entirety.

[0012] In the present invention, the term "gene" includes double-stranded DNA including human genomic DNA, single-stranded DNA (positive strand) including cDNA, single-stranded DNA (complementary strand) having a sequence complementary to the positive strand, and fragments thereof, and refers to DNA in which some biological information is contained in the sequence information of the bases that make up the DNA. Furthermore, the "gene" in question includes not only "genes" represented by a specific base sequence, but also nucleic acids encoding their homologues (i.e., homologs or orthologs), variants such as genetic polymorphisms, and derivatives. The names of the genes disclosed herein are based on the official symbols listed in NCBI ([www.ncbi.nlm.nih.gov / ]).

[0013] In the present invention, the term "expression product" of a gene encompasses both transcription products and translation products of the gene. A "transcription product" is RNA generated by transcription from a gene (DNA), and a "translation product" refers to a protein encoded by the gene that is translated and synthesized based on the RNA.

[0014] In the present invention, "atopic dermatitis" refers to a disease whose main pathogenic factor is an itchy eczema that repeatedly worsens and remits, and many of its patients are said to have a predisposition to atopy. Examples of atopic predisposition include i) a family history or medical history (one or more of the following diseases: bronchial asthma, allergic rhinitis / conjunctivitis, and atopic dermatitis), or ii) a predisposition to easily produce IgE antibodies.

[0015] In the present invention, "severity of atopic dermatitis" means the degree of progression of atopic dermatitis, and is classified according to the condition of the rash, for example, as no symptoms, slight, mild (mild), moderate (moderate), or severe (severe). The characteristics of the rash include erythema, edema / infiltration / papules (solid, serous), exudate / crust, scarring, lichenification, dryness, pruritus, prurigo nodules, scales (pityriasis, foliaceous, membranous, etc.), blisters, pustules, erosions, and ulcers. The severity can be classified into categories such as Eczema Area and Severity. Index("EASI"<Exp Dermatol, 2001; 10: 11-18.> ), Investigator's Global Assessment("IGA"<J Am Acad Dermatol, 2016; 74: 288-94.> ), as well as the atopic dermatitis severity classification by the Atopic Dermatitis Severity Classification Committee of the Japanese Dermatological Association (JDAS 2001; 111: 2023-2033, JDAS 1998; 108: 1491-1496.), the Severity Scoring of Atopic Dermatitis (SCORAD)<Dermatology, 1993; 186: 23-31.> etc. are known.

[0016] For example, the EASI is a score ranging from 0 to 72, calculated based on the evaluation sites of the head and neck, trunk, upper limbs, and lower limbs, and the scores for four symptoms (erythema, edema / infiltration / papule, excoriation, and lichenification) at each evaluation site, as well as the percentage (%) of the area of ​​the above four symptoms relative to the entire evaluation site. In IGA, the assessor, a physician or researcher, comprehensively evaluates the condition of the rash on the whole body or on a specific evaluation site and assigns a five-point rating: "no symptoms," "mild," "mild," "moderate," or "severe." A six-point rating, including "very severe," may also be used. IGA scores can be converted to an ordinal scale, such as "no symptoms = 0," "mild = 1," "mild = 2," "moderate = 3," "severe = 4," and "very severe = 5."

[0017] In the detection of the severity of atopic dermatitis according to the present invention, "no symptoms," "mild," "moderate," and "severe" can be detected, but it is preferable to detect "no symptoms," "mild," and "moderate." In the present invention, "mild" refers to a condition mainly characterized by dryness, mild erythema, and scales, and corresponds to an EASI score of more than 0 and less than 6, or an IGA score of 2. "Moderate" refers to a condition mainly characterized by moderate to severe erythema, scales, a few papules, and excoriations, and corresponds to an EASI score of 6 or more and less than 23, or an IGA score of 3. "Severe" refers to a condition mainly characterized by erythema accompanied by severe swelling / edema / infiltration or lichenification, multiple papules, severe scaling, crusting, small blisters, erosions, numerous excoriations, and prurigo nodules, and corresponds to an EASI score of 23 or more and 72 or an IGA score of 4. "No symptoms" refers to remission or near remission with no symptoms, at the same level as a healthy person. In addition, when assessing "no symptoms," "mild," "moderate," and "severe" using the EASI score or IGA score, the range of scores used as the basis for each assessment is not limited to the above and can be determined as appropriate.

[0018] In the present invention, "detection" of the severity of atopic dermatitis means clarifying the severity of atopic dermatitis, and can be expressed in other words as examination, measurement, judgment, or evaluation support. Note that, in the present invention, the terms "detection," "examination," "measurement," "judgment," or "evaluation" do not include a diagnosis of the severity of atopic dermatitis by a doctor.

[0019] As shown in the Examples below, 29 atopic dermatitis patients, including 14 healthy individuals, 18 diagnosed as mildly ill (score: greater than 0 and less than 6) and 11 as moderately ill (score: 6 or greater and less than 23) according to the EASI severity scale, underwent RNA expression analysis using the following steps 1) to 2). As a result, the expression of five genes, CAPN1, GRN, NCOR2, PLP2, and PPP1R9B, increased in a severity-dependent manner. 1) Obtain data on the expression level (read count values) of RNA extracted from SSLs of 14 healthy individuals and 29 patients (adults) with atopic dermatitis. 2) The read count value is converted into an RPM value corrected for the difference in the total number of reads between samples, and the RPM+1 value is added by an integer 1 to this value, which is converted into a logarithmic value with base 2 (Log2(RPM+1) value). Based on this value, a Tukey test is performed between the three groups (healthy subjects, mild patients, and moderate patients), and genes with a p-value of less than 0.1 are selected in all of the following groups: healthy subjects vs. mild patients, mild patients vs. moderate patients, and healthy subjects vs. moderate patients. Here, the "p-value" indicates the probability that a statistical value that is more extreme than the statistical value actually calculated from the data under the null hypothesis will be observed in a statistical test. Therefore, the smaller the "p-value," the more significant the difference between the compared objects.

[0020] In addition, 29 atopic dermatitis patients, including 14 healthy individuals and 17 individuals diagnosed with mild symptoms and 12 individuals judged to have moderate symptoms based on a comprehensive assessment of the facial IGA on a five-point scale of 0 (no symptoms), 1 (mild), 2 (mild), 3 (moderate), and 4 (severe), were subjected to RNA expression analysis using steps 1) to 2) as described above. The results showed that the expression of three genes, LOC146880, PPP1R12C, and SYVN1, increased in a severity-dependent manner, and that the expression of SLC31A1 decreased in a severity-dependent manner.

[0021] Therefore, a gene or its expression product selected from a group of nine genes consisting of CAPN1, NCOR2, PPP1R9B, PLP2, GRN, PPP1R12C, LOC146880, SLC31A1, and SYVN1 can be used as a detection marker for the severity of atopic dermatitis. Each of these nine genes can be used alone as a marker for detecting the severity of atopic dermatitis, but two or more of them can also be used in combination. Preferably, to detect the severity of atopic dermatitis based on EASI, detection markers are selected from five of the nine genes, namely, CAPN1, NCOR2, PPP1R9B, PLP2, and GRN, and to detect the severity of atopic dermatitis based on IGA, detection markers are selected from four of the nine genes, namely, PPP1R12C, LOC146880, SLC31A1, and SYVN1. Furthermore, these nine genes are novel markers for determining the severity of atopic dermatitis, the relationship of which has not been reported to the severity of atopic dermatitis until now.

[0022] The above-mentioned genes that can serve as markers for detecting the severity of atopic dermatitis (hereinafter also referred to as "target genes") also include genes having a nucleotide sequence substantially identical to that of the DNA constituting the gene, as long as they can serve as biomarkers for detecting the severity of atopic dermatitis. Here, "substantially identical" means, for example, that when searched using the homology calculation algorithm NCBI BLAST under the conditions of expectation value = 10; gaps allowed; filtering = ON; match score = 1; mismatch score = -3, the nucleotide sequence has 90% or more, preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more identity with the nucleotide sequence of the DNA constituting the gene.

[0023] The method for detecting the severity of atopic dermatitis of the present invention includes a step of measuring the expression level of a target gene, in one embodiment, at least one gene or its expression product selected from a group of nine genes consisting of CAPN1, NCOR2, PPP1R9B, PLP2, GRN, PPP1R12C, LOC146880, SLC31A1, and SYVN1, in a biological sample collected from a subject.

[0024] In the method for detecting the severity of atopic dermatitis of the present invention, the sex, age, race, etc. of the subject are not particularly limited, and may include anyone from infants to the elderly. Preferably, the subject is a human who needs or desires to have the severity of atopic dermatitis detected. For example, the subject is a human who has developed atopic dermatitis, a human who is suspected of developing atopic dermatitis, or a human who is genetically predisposed to atopic dermatitis.

[0025] The biological sample used in the present invention may be any tissue or biological material in which the expression of the gene of the present invention changes depending on the severity of atopic dermatitis. Specific examples include organs, skin, blood, urine, saliva, sweat, stratum corneum, skin surface lipids (SSL), tissue exudates, and other body fluids; serum and plasma prepared from blood; and feces and hair. Preferred examples include skin, stratum corneum, or skin surface lipids (SSL), and more preferred examples include skin, stratum corneum, or skin surface lipids (SSL). The site from which SSL is collected is not particularly limited and may include skin from any part of the body, such as the head, face, neck, trunk, hands, or feet. Sites with high sebum secretion, such as the skin of the head or face, are preferred, and facial skin is more preferred. Furthermore, the site from which SSL is collected may be either a rash area with atopic dermatitis or a non-rash area without atopic dermatitis. Preferably, the site is a rash area or a non-rash area adjacent to the rash area. Here, "near the rash area" refers to an area within 10 cm adjacent to the rash area.

[0026] Here, "skin surface lipids (SSL)" refers to the fat-soluble fraction present on the surface of the skin, and is sometimes called sebum. Generally, SSL mainly contains secretions from exocrine glands such as sebaceous glands in the skin, and is present on the skin surface in the form of a thin layer that covers the skin surface. SSL contains RNA expressed in skin cells (see Patent Document 1). In addition, in this specification, unless otherwise specified, "skin" is a general term for the region including the stratum corneum, epidermis, dermis, hair follicles, and tissues such as sweat glands, sebaceous glands, and other glands.

[0027] Any method commonly used for recovering or removing SSL from skin can be used to collect SSL from a subject's skin. Preferably, SSL absorbent materials, SSL adhesive materials, or devices for scraping SSL from skin, as described below, can be used. The SSL absorbent material or SSL adhesive material can be any material that has affinity for SSL, including polypropylene and pulp. More detailed examples of procedures for collecting SSL from skin include absorbing SSL into sheet-like materials such as oil blotting paper or oil blotting film, adhering SSL to glass plates or tape, or scraping SSL off with a spatula or scraper. To improve SSL adsorption, SSL absorbent materials pre-soaked with a highly lipid-soluble solvent may be used. However, SSL absorbent materials preferably contain low amounts of highly water-soluble solvents or moisture, since the presence of highly water-soluble solvents or moisture inhibits SSL adsorption. It is preferable to use SSL absorbent materials in a dry state.

[0028] The RNA-containing SSL collected from a subject may be stored for a certain period of time. To minimize degradation of the RNA contained in the collected SSL, it is preferable to store the collected SSL under low-temperature conditions as soon as possible after collection. The temperature conditions for storing the RNA-containing SSL in the present invention may be 0°C or lower, preferably -20±20°C to -80±20°C, more preferably -20±10°C to -80±10°C, even more preferably -20±20°C to -40±20°C, even more preferably -20±10°C to -40±10°C, even more preferably -20±10°C, and even more preferably -20±5°C. The period for storing the RNA-containing SSL under low-temperature conditions is not particularly limited, but is preferably 12 months or less, for example, 6 hours to 12 months, more preferably 6 months or less, for example, 1 day to 6 months, even more preferably 3 months or less, for example, 3 days to 3 months.

[0029] In the present invention, objects for measuring the expression level of a target gene or its expression product include cDNA artificially synthesized from RNA, DNA encoding that RNA, a protein encoded by that RNA, a molecule that interacts with the protein, a molecule that interacts with that RNA, or a molecule that interacts with that DNA. Here, molecules that interact with RNA, DNA, or protein include DNA, RNA, proteins, polysaccharides, oligosaccharides, monosaccharides, lipids, fatty acids, and their phosphorylations, alkylations, and sugar adducts, as well as complexes of any of the above. Furthermore, the expression level comprehensively refers to the expression amount and activity of the gene or expression product.

[0030] In a preferred embodiment of the method of the present invention, SSL is used as the biological sample, in which case the expression level of RNA contained in the SSL is analyzed, specifically by converting the RNA into cDNA by reverse transcription, and then measuring the cDNA or its amplification product. RNA can be extracted from SSL using methods commonly used for extracting or purifying RNA from biological samples, such as the phenol / chloroform method, the AGPC (acid guanidinium thiocyanate-phenol-chloroform extraction) method, methods using columns such as TRIzol (registered trademark), RNeasy (registered trademark), or QIAzol (registered trademark), methods using special silica-coated magnetic particles, methods using Solid Phase Reversible Immobilization magnetic particles, and extraction using commercially available RNA extraction reagents such as ISOGEN.

[0031] For the reverse transcription, a primer targeting the specific RNA to be analyzed may be used, but for more comprehensive nucleic acid storage and analysis, random primers are preferably used. A general reverse transcriptase or reverse transcription reagent kit can be used for the reverse transcription. Highly accurate and efficient reverse transcriptases or reverse transcription reagent kits are preferably used, such as M-MLV Reverse Transcriptase and its variants, or commercially available reverse transcriptases or reverse transcription reagent kits, such as the PrimeScript® Reverse Transcriptase series (Takara Bio Inc.) and the SuperScript® Reverse Transcriptase series (Thermo Scientific). SuperScript® III Reverse Transcriptase and SuperScript® VILO cDNA Synthesis kit (both Thermo Scientific) are preferably used. The temperature of the extension reaction in the reverse transcription is preferably adjusted to 42°C±1°C, more preferably 42°C±0.5°C, and even more preferably 42°C±0.25°C, while the reaction time is preferably adjusted to 60 minutes or more, more preferably 80 to 120 minutes.

[0032] When targeting RNA, cDNA, or DNA, the method for measuring the expression level can be selected from the following: PCR using DNA that hybridizes to these as a primer, nucleic acid amplification methods such as real-time RT-PCR, multiplex PCR, SmartAmp, and LAMP; hybridization methods using nucleic acids that hybridize to these as probes (DNA chips, DNA microarrays, dot blot hybridization, slot blot hybridization, Northern blot hybridization, etc.); methods for determining base sequences (sequencing); or a combination of these.

[0033] In PCR, a primer pair targeting a specific DNA to be analyzed may be used to amplify only that specific DNA, or multiple primer pairs may be used to simultaneously amplify multiple specific DNAs. Preferably, the PCR is multiplex PCR. Multiplex PCR is a method in which multiple gene regions are simultaneously amplified by simultaneously using multiple primer pairs in a PCR reaction system. Multiplex PCR can be performed using a commercially available kit (e.g., Ion AmpliSeq Transcriptome Human Gene Expression Kit; Life Technologies Japan, Inc., etc.). The temperatures for the annealing and extension reactions in the PCR depend on the primers used and cannot be generalized; however, when using the multiplex PCR kit described above, the temperatures are preferably 62°C ± 1°C, more preferably 62°C ± 0.5°C, and even more preferably 62°C ± 0.25°C. Therefore, in the PCR, the annealing and extension reactions are preferably carried out in one step. The time for the annealing and extension reaction steps can be adjusted depending on the size of the DNA to be amplified, but is preferably 14 to 18 minutes. The conditions for the denaturation reaction in the PCR can be adjusted depending on the DNA to be amplified, but are preferably 95 to 99°C for 10 to 60 seconds. Reverse transcription and PCR at the temperatures and times described above can be carried out using a thermal cycler commonly used for PCR.

[0034] The purification of the reaction product obtained by the PCR is preferably carried out by size separation of the reaction product. By size separation, the target PCR reaction product can be separated from primers and other impurities contained in the PCR reaction solution. Size separation of DNA can be carried out using, for example, a size separation column, a size separation chip, magnetic beads usable for size separation, etc. Preferred examples of magnetic beads usable for size separation include Solid Phase Reversible Immobilization (SPRI) magnetic beads such as Ampure XP.

[0035] The purified PCR reaction product may be further processed as necessary for subsequent quantitative analysis. For example, for DNA sequencing, the purified PCR reaction product may be prepared in an appropriate buffer solution, the PCR primer region contained in the PCR-amplified DNA may be cleaved, or an adapter sequence may be further added to the amplified DNA. For example, the purified PCR reaction product may be prepared in a buffer solution, and the amplified DNA may be subjected to PCR primer sequence removal and adapter ligation. The resulting reaction product may then be amplified as necessary to prepare a library for quantitative analysis. These operations may be performed, for example, using the 5x VILO RT Reaction Mix included in the SuperScript® VILO cDNA Synthesis kit (Life Technologies Japan, Inc.), and the 5x Ion AmpliSeq HiFi Mix and Ion AmpliSeq Transcriptome Human Gene Expression Kit (Life Technologies Japan, Inc.). This can be done using the Gene Expression Core Panel and following the protocol provided with each kit.

[0036] When measuring the expression level of a target gene or a nucleic acid derived therefrom using Northern blot hybridization, for example, probe DNA is first labeled with a radioisotope, a fluorescent substance, or the like, and then the resulting labeled DNA is hybridized with RNA derived from a biological sample that has been transferred to a nylon membrane or the like in a standard manner. The resulting double strand of labeled DNA and RNA is then measured by detecting a signal derived from the label.

[0037] When measuring the expression level of a target gene or a nucleic acid derived therefrom using RT-PCR, for example, cDNA is first prepared from RNA derived from a biological sample according to standard methods, and then a pair of primers (a positive strand that binds to the cDNA (-strand) and a reverse strand that binds to the + strand) prepared so that the target gene of the present invention can be amplified using this as a template are hybridized to the cDNA. PCR is then performed according to standard methods, and the resulting amplified double-stranded DNA is detected. The amplified double-stranded DNA can be detected by a method such as detecting labeled double-stranded DNA produced by performing the above-mentioned PCR using primers that have been labeled in advance with RI, a fluorescent substance, or the like.

[0038] When measuring the expression level of a target gene or a nucleic acid derived therefrom using a DNA microarray, for example, an array having at least one type of nucleic acid (cDNA or DNA) derived from the target gene of the present invention immobilized on a support is used, labeled cDNA or cRNA prepared from mRNA is bound to the microarray, and the label on the microarray is detected, thereby measuring the expression level of mRNA. The nucleic acid immobilized on the array may be any nucleic acid that hybridizes specifically (i.e., substantially only to the target nucleic acid) under stringent conditions. For example, it may be a nucleic acid having the entire sequence of the target gene of the present invention, or a nucleic acid consisting of a partial sequence. Here, a "partial sequence" refers to a nucleic acid consisting of at least 15 to 25 bases. Typical stringent conditions include washing conditions of approximately 1×SSC, 0.1% SDS, and 37°C. More stringent hybridization conditions include approximately 0.5×SSC, 0.1% SDS, and 42°C. Even more stringent hybridization conditions include approximately 0.1×SSC, 0.1% SDS, and 65°C. Hybridization conditions are described in, for example, J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001).

[0039] When measuring the expression level of a target gene or a nucleic acid derived therefrom by sequencing, for example, analysis can be performed using a next-generation sequencer (e.g., Ion S5 / XL system, Life Technologies Japan, Inc.). RNA expression can be quantified based on the number of reads (read count) generated by sequencing.

[0040] The probes or primers used in the above measurements, i.e., primers for specifically recognizing and amplifying the target gene of the present invention or a nucleic acid derived therefrom, or probes for specifically detecting the RNA or a nucleic acid derived therefrom, fall into this category, and can be designed based on the nucleotide sequence constituting the target gene. Here, "specifically recognize" means that the detected substance or product can be determined to be the gene or a nucleic acid derived therefrom, such that, for example, in Northern blotting, substantially only the target gene of the present invention or a nucleic acid derived therefrom can be detected, or, for example, in RT-PCR, substantially only the nucleic acid is amplified. Specifically, DNA consisting of the base sequence constituting the target gene of the present invention or an oligonucleotide containing a certain number of nucleotides complementary to its complementary strand can be used. Here, "complementary strand" refers to one strand of a double-stranded DNA consisting of A:T (U in the case of RNA) and G:C base pairs, while the other strand is complementary to the other strand. Furthermore, "complementary" does not necessarily mean a perfectly complementary sequence over a certain number of consecutive nucleotides, but rather means that the sequence has a base sequence identity of preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 98% or more. The base sequence identity can be determined using an algorithm such as BLAST. When used as a primer, such an oligonucleotide may be capable of specific annealing and chain elongation, and typically has a chain length of, for example, 10 or more bases, preferably 15 or more bases, more preferably 20 or more bases, and for example, 100 or less bases, preferably 50 or less bases, more preferably 35 or less bases. When used as a probe, it is sufficient to be capable of specific hybridization, and an oligonucleotide having at least a partial or complete sequence of DNA (or its complementary strand) consisting of the base sequence constituting the target gene of the present invention, and having a chain length of, for example, 10 or more bases, preferably 15 or more bases, and for example, 100 or less bases, preferably 50 or less bases, more preferably 25 or less bases, is used. Here, "oligonucleotide" can be DNA or RNA, and can be either synthetic or natural. Furthermore, the probe used for hybridization is usually labeled.

[0041] Furthermore, when measuring the translation product (protein) of the target gene of the present invention, a molecule that interacts with the protein, a molecule that interacts with RNA, or a molecule that interacts with DNA, methods such as protein chip analysis, immunoassays (e.g., ELISA, etc.), mass spectrometry (e.g., LC-MS / MS, MALDI-TOF / MS), the one-hybrid method (PNAS 100, 12271-12276 (2003)), or the two-hybrid method (Biol. Reprod. 58, 302-311 (1998)) can be used, and can be appropriately selected depending on the subject. For example, when a protein is used as the measurement target, the measurement is carried out by contacting an antibody against the expression product of the present invention with a biological sample, detecting the polypeptide in the sample that binds to the antibody, and measuring its level. For example, in the Western blot method, the above-mentioned antibody is used as the primary antibody, and then an antibody that binds to the primary antibody and is labeled with a radioisotope, a fluorescent substance, an enzyme, or the like is used as the secondary antibody, and the primary antibody is labeled, and the signal derived from this label is measured using a radiation measuring instrument, a fluorescence detector, or the like. The antibody against the translation product may be a polyclonal antibody or a monoclonal antibody. These antibodies can be produced according to known methods. Specifically, polyclonal antibodies can be obtained according to standard methods by immunizing a non-human animal such as a rabbit with a protein expressed in E. coli or the like and purified according to standard methods, or by synthesizing a partial polypeptide of the protein according to standard methods, and then extracting the antibody from the serum of the immunized animal. On the other hand, monoclonal antibodies can be obtained from hybridoma cells prepared by immunizing a non-human animal such as a mouse with a protein expressed and purified in Escherichia coli or a partial polypeptide of the protein according to a conventional method, and fusing the resulting spleen cells with myeloma cells. Monoclonal antibodies can also be produced using phage display (Griffiths, AD; Duncan, AR, Current Opinion in Biotechnology, Volume 9, Number 1, February 1998, pp. 102-108(7)).

[0042] Thus, the expression level of the target gene of the present invention or its expression product in a biological sample collected from a subject is measured, and the severity of atopic dermatitis is detected based on the expression level. Specifically, the detection is carried out by comparing the measured expression level of the target gene of the present invention or its expression product with a control level. When analyzing the expression levels of multiple target genes by sequencing, as described above, it is preferable to use as indicators the read count values, which are expression level data; the RPM values ​​obtained by correcting the read count values ​​for differences in the total number of reads between samples; the RPM values ​​converted to base 2 logarithms (Log2RPM values) or base 2 logarithms obtained by adding an integer 1 (Log2(RPM+1) values); or count values ​​corrected using DESeq2 (Love MI et al. Genome Biol. 2014) (Normalized count values) or base 2 logarithms obtained by adding an integer 1 (Log2(count+1) values). Furthermore, values ​​calculated using common quantitative values ​​for RNA-seq, such as fragments per kilobase of exon per million reads mapped (FPKM), reads per kilobase of exon per million reads mapped (RPKM), or transcripts per million (TPM), may also be used. Furthermore, signal values ​​obtained by microarray analysis and their corrected values ​​may also be used. Furthermore, when analyzing only a specific target gene by RT-PCR or the like, it is preferable to convert the expression level of the target gene into a relative expression level based on the expression level of a housekeeping gene, or to quantify the absolute copy number (absolute quantification) using a plasmid containing the target gene region. The copy number obtained by digital PCR may also be used. Here, the "control level" refers to, for example, the expression level of the target gene or its expression product in healthy individuals when detecting mildly ill patients, or in mildly ill patients when detecting moderately ill patients. The expression levels in healthy individuals or mildly ill patients may be statistical values ​​(e.g., average values) of the expression levels of the gene or its expression product measured from a group of healthy individuals or mildly ill patients. When there are multiple target genes, it is preferable to determine the reference expression level for each gene or its expression product.

[0043] In addition, the severity of atopic dermatitis in the present invention can also be detected by measuring an increase / decrease in the expression level of the target gene of the present invention or its expression product. In this case, the expression level of the target gene or its expression product in a biological sample derived from a subject is compared with a cutoff value (reference value) for each gene or its expression product. The cutoff value can be determined appropriately based on statistical values ​​such as the average value and standard deviation of the expression levels obtained in advance from reference data obtained from healthy individuals, mildly affected patients, moderately affected patients, and severely affected patients.

[0044] Furthermore, using measured values ​​of the expression levels of target genes or their expression products from patients with atopic dermatitis of different severity and those from healthy individuals, a discriminant (prediction model) can be constructed to separate patient groups with different severity levels (e.g., mild, moderate, and severe patients) from healthy individuals (asymptomatic), and the severity of atopic dermatitis can be detected using the discriminant. Specifically, using measured values ​​of the expression levels of target genes or their expression products from patients with atopic dermatitis of different severity and those from healthy individuals as training samples, a discriminant (prediction model) can be constructed to separate patient groups with different severity levels (e.g., mild, moderate, and severe patients) from healthy individuals (asymptomatic), and a cutoff value (reference value) for distinguishing between patient groups with different severity levels can be determined based on the discriminant. In constructing the discriminant, dimensionality reduction can be performed using principal component analysis (PCA), and the principal components can be used as explanatory variables. The level of the target gene or its expression product is then similarly measured from a biological sample collected from the subject, the obtained measurement value is substituted into the discriminant, and the result obtained from the discriminant is compared with a reference value, thereby enabling detection of the severity of atopic dermatitis in the subject.

[0045] As an algorithm for constructing a discriminant, a known algorithm such as an algorithm used in machine learning can be used. Examples of machine learning algorithms include random forest and linear kernel support vector machine (SVM). Examples of suitable predictive models include neural networks (NNNs), generalized linear models, regularized linear discriminant analysis (Regularized Linear Discriminant Analysis (RBF)), neural networks with an rbf kernel, generalized linear models, regularized linear discriminant analysis (Regularized Logistic Regression), and so on. Verification data is input into the constructed predictive model to calculate predicted values, and the model whose predicted values ​​best match the actual measured values, for example, the model with the highest accuracy, can be selected as the optimal predictive model. Furthermore, recall, precision, and the F-value, which is the harmonic mean of these, can be calculated from the predicted and actual measured values, and the model with the highest F-value can be selected as the optimal predictive model.

[0046] The method for determining the cutoff value (reference value) is not particularly limited, and can be determined according to known techniques. For example, it can be determined from an ROC (Receiver Operating Characteristic Curve) curve created using a discriminant. In an ROC curve, the vertical axis plots the probability of a positive result in a positive patient (sensitivity), and the horizontal axis plots the value obtained by subtracting the probability of a negative result in a negative patient (specificity) from 1 (false positive rate). Regarding the "true positive (sensitivity)" and "false positive (1-specificity)" shown in the ROC curve, the value (Youden index) at which "true positive (sensitivity)" - "false positive (1-specificity)" is maximized can be used as the cutoff value (reference value).

[0047] The test kit for detecting the severity of atopic dermatitis of the present invention contains test reagents for measuring the expression level of the target gene of the present invention or its expression product in a biological sample isolated from a patient. Specific examples include reagents for nucleic acid amplification or hybridization, including oligonucleotides (e.g., PCR primers) that specifically bind (hybridize) to the target gene of the present invention or a nucleic acid derived therefrom, and reagents for immunological measurements, including antibodies that recognize the expression product (protein) of the target gene of the present invention. The oligonucleotides, antibodies, etc. included in the kit can be obtained by known methods, as described above. In addition to the above-mentioned antibodies and nucleic acids, the test kit may also include labeling reagents, buffer solutions, color-developing substrates, secondary antibodies, blocking agents, equipment necessary for the test, control reagents used as positive and negative controls, and tools for collecting biological samples (e.g., oil blotting films for collecting SSL).

[0048] In relation to the above-described embodiment, the present invention further discloses the following aspects. <1> A method for detecting the severity of atopic dermatitis in a subject, comprising a step of measuring the expression level of at least one gene or its expression product selected from a group of nine genes consisting of CAPN1, NCOR2, PPP1R9B, PLP2, GRN, PPP1R12C, LOC146880, SLC31A1 and SYVN1 in a biological sample collected from the subject. <2> The severity is based on the EASI score or IGA score. <1> How to do it. <3> The severity is based on the EASI score, and the measurement of the expression level is the measurement of the expression level of at least one gene selected from a group of five genes consisting of CAPN1, NCOR2, PPP1R9B, PLP2, and GRN, or its expression product. <2> How to do it. <4> the severity is based on the IGA score, and the measurement of the expression level is the measurement of the expression level of at least one gene selected from a group of four genes consisting of PPP1R12C, LOC146880, SLC31A1, and SYVN1, or its expression product; <2> How to do it. <5> The severity is one or more of the following: no symptoms, mild symptoms, and moderate symptoms. <1> ~ <4> Either way. <6> The expression level of a gene or its expression product is measured by measuring the expression amount of mRNA. <1> ~ <5> Either way. <7> The gene or its expression product is RNA contained in the lipids on the skin surface of the subject. <1> ~ <6> Either way. <8> the measured expression levels are compared with reference values ​​for each of the genes or their expression products to detect the severity of atopic dermatitis; <1> ~ <7> Either way. <9> Use of at least one gene or its expression product selected from a group of nine genes consisting of CAPN1, NCOR2, PPP1R9B, PLP2, GRN, PPP1R12C, LOC146880, SLC31A1 and SYVN1 derived from a biological sample collected from a subject as a marker for the severity of atopic dermatitis. <10> Use of at least one gene or its expression product selected from a group of five genes consisting of CAPN1, NCOR2, PPP1R9B, PLP2, and GRN, and the severity is based on the EASI score. <9> Use of. <11> Use of at least one gene or its expression product selected from a group of four genes consisting of PPP1R12C, LOC146880, SLC31A1, and SYVN1, wherein the severity is based on an IGA score. <9> Use of. <12> The gene or its expression product is mRNA contained in lipids on the skin surface collected from the subject. <9> ~ <11> Use of either. <13> An oligonucleotide that specifically hybridizes with the gene or a nucleic acid derived therefrom, or an antibody that recognizes the expression product of the gene, <1> ~ <8> A test kit for detecting the severity of atopic dermatitis, which is used in any of the methods above. <14> A marker for detecting the severity of atopic dermatitis, consisting of at least one gene or its expression product selected from a group of nine genes consisting of CAPN1, NCOR2, PPP1R9B, PLP2, GRN, PPP1R12C, LOC146880, SLC31A1 and SYVN1. [Example]

[0049] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples. Example 1: Detection of the severity of atopic dermatitis using RNA extracted from SSL 1) Diagnosis of atopic dermatitis patients and SSL collection The subjects were 14 healthy individuals (25-57 years old, male) and 29 adults with atopic dermatitis (AD) (23-56 years old, male). Patients with atopic dermatitis were diagnosed by a dermatologist as having mild or moderate atopic dermatitis. The EASI score (Hanifin et al. Exp Dermatol. 10, 2001) was used for diagnosis. Patients with a score greater than 0 but less than 6 were considered to have mild atopic dermatitis, and those with a score greater than 6 but less than 23 were considered to have moderate atopic dermatitis (Chopra et al. Br J Dermatol. 177, 2017). As a result, 18 patients had mild atopic dermatitis and 11 had moderate atopic dermatitis. Furthermore, a comprehensive evaluation was performed on the faces of the above-mentioned atopic dermatitis patients using an IGA score on a five-point scale: 0 (no symptoms), 1 (very slight), 2 (mild), 3 (moderate), and 4 (severe). As a result, 17 patients had mild symptoms with a score of 2, and 12 patients had moderate symptoms with a score of 3. After collecting sebum from the entire face of each subject using an oil-blotting film, the oil-blotting film was transferred to a vial and stored at -80°C for approximately one month until it was used for RNA extraction.

[0050] 2) RNA preparation and sequencing The oil blotting film (1) above was cut to an appropriate size, and RNA was extracted using QIAzol Lysis Reagent (Qiagen) according to the attached protocol. The extracted RNA was reverse-transcribed at 42°C for 90 minutes using the SuperScript VILO cDNA Synthesis kit (Life Technologies Japan, Inc.) to synthesize cDNA. The random primers included with the kit were used as primers for the reverse transcription reaction. A library containing DNA derived from the 20802 gene was prepared from the resulting cDNA by multiplex PCR. Multiplex PCR was performed using the Ion AmpliSeq Transcriptome Human Gene Expression Kit (Life Technologies Japan, Inc.) under the following conditions: 99°C, 2 minutes → (99°C, 15 seconds → 62°C, 16 minutes) × 20 cycles → 4°C hold. The resulting PCR products were purified using Ampure XP (Beckman Coulter, Inc.), followed by buffer reconstitution, primer digestion, adapter ligation, purification, and amplification to prepare a library. The prepared library was loaded onto an Ion 540 chip and sequenced using an Ion S5 / XL system (Life Technologies Japan, Inc.).

[0051] 3) Data analysis 1: Genes with varying severity depending on the EASI score i) Usage Data The RNA expression level data (read count values) from the subjects measured in 2) above were converted to RPM values ​​corrected for differences in the total number of reads between samples. However, only 7,429 genes for which non-missing expression level data was obtained in more than 90% of all samples were used in the following analysis. To approximate the RPM values, which follow a negative binomial distribution, to a normal distribution, the base 2 logarithm (Log2(RPM+1) value) obtained by adding an integer 1 was used. Next, the samples were divided into three groups: healthy subjects, mildly affected patients, and moderately affected patients, according to the severity classification based on the EASI score evaluated in 1) above.

[0052] ii) RNA expression analysis Based on the SSL-derived RNA expression levels (Log2(RPM+1) values) of healthy, mild, and moderately affected individuals measured in i) above, a Tukey's test was performed among the three groups, and genes with p values ​​of less than 0.1 were selected for all comparisons between healthy and mildly affected individuals, mild and moderately affected individuals, and healthy and moderately affected individuals. As a result, the expression of five genes, CAPN1, GRNNCOR2, PLP2, and PPP1R9B, increased in a severity-dependent manner (Table 1).

[0053] [Table 1-1]

[0054] [Table 1-2]

[0055] 4) Data analysis 2: Genes that vary depending on severity classification by facial comprehensive assessment (IGA score) i) Usage Data The RNA expression data (read count values) from the subjects measured in 2) above were converted to RPM values ​​corrected for differences in the total number of reads between samples. Only 7,429 genes for which non-missing expression data was obtained in 90% or more of the total samples were used in the following analysis. To approximate the RPM values, which follow a negative binomial distribution, to a normal distribution, the base 2 logarithm (Log2(RPM+1) value) obtained by adding an integer 1 was used. Next, the samples were divided into three groups: healthy subjects, mildly affected patients, and moderately affected patients, according to the severity classification based on the facial global assessment (IGA score) evaluated in 1) above.

[0056] ii) RNA expression analysis Based on the SSL-derived RNA expression levels (Log2(RPM+1) values) measured in i) above for healthy subjects, mildly affected patients, and moderately affected patients, a Tukey's test was performed among the three groups, and genes with p values ​​of less than 0.1 were selected for all comparisons between healthy subjects and mildly affected patients, mildly affected patients and moderately affected patients, and healthy subjects and moderately affected patients. As a result, the expression of three genes, LOC146880, PPP1R12C, and SYVN1, increased in a severity-dependent manner, and SLC31A1 decreased in a severity-dependent manner (Table 2).

[0057] [Table 2-1]

[0058] [Table 2-2]

Claims

1. A method for detecting the severity of atopic dermatitis in a subject, comprising measuring the expression level of mRNA of at least one gene selected from a group of five genes consisting of CAPN1, NCOR2, PPP1R9B, PLP2, and GRN in lipids on the skin surface collected from the entire face of the subject, wherein the severity is based on the EASI (Eczema Area and Severity Index) score, and comparing the measured expression level of the subject with reference values ​​for the expression level of mRNA of each of the genes, which reference values ​​are used to distinguish between atopic dermatitis patient groups with different severity levels; A higher measured value of the expression level of the subject than the reference value indicates a higher severity of the disease. method.

2. A method for detecting the severity of atopic dermatitis in a subject, comprising measuring the expression level of mRNA of at least one gene selected from a group of four genes consisting of PPP1R12C, LOC146880, SLC31A1, and SYVN1 in lipids on the skin surface collected from the entire face of the subject, wherein the severity is based on an IGA (Investigator's Global Assessment) score, and comparing the measured expression level of the subject with reference values ​​for the expression level of mRNA of each of the genes, which reference values ​​are used to distinguish between atopic dermatitis patient groups with different severity levels; (1) when the gene is PPP1R12C, LOC146880, or SYVN1, a higher measured value of the expression level of the subject than the reference value indicates a higher severity; (2) When the gene is SLC31A1, a smaller measured value of the expression level of the subject than the reference value indicates a higher severity. method.

3. The method according to claim 1 or 2, wherein the severity is one or more selected from the group consisting of no symptoms, mild symptoms, and moderate symptoms.

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