Method for detecting seriousness of atopic dermatitis

Abstract

To provide a method for measuring a gene for detecting seriousness of atopic dermatitis or an expression product thereof.SOLUTION: A method for measuring a gene in a subject or an expression product thereof includes a step of measuring an expression level of at least one gene selected from 16 kinds of gene group consisting of C1QB, DIAPH1, LCE1A, CTSV, CTSZ, KRT72, SPINK5, KRT23, KLK5, LCE1B, SPRR1A, PKP1, KRT74, APRT, KRT25 and S100B or an expression product thereof regarding a biological sample collected from the subject in order to detect seriousness of atopic dermatitis in the subject.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 mainly occurs in people with atopic predisposition. Typical symptoms of atopic dermatitis include chronic and recurrent itching, rash, erythema, etc. occurring on both sides of the body, as well as keratinization failure, impaired barrier function, dry skin, etc. Most cases of atopic dermatitis occur in infants and young children, and tend to improve with age, but in recent years, adult-type 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, a test method using blood TARC (thymus and activation-regulated chemokine) value and SCCA2 (squamous cell carcinoma antigen 2) value as indicators has been used as a method for objectively judging the severity of atopic dermatitis. These are used as indicators that sensitively reflect the progress of the disease, since the amount present in the blood increases depending on the severity 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 plans (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] On the other hand, techniques have been developed to investigate the present and future physiological state of the human body by analyzing nucleic acids such as DNA and RNA in biological samples. Analysis using nucleic acids has the advantages that comprehensive analysis methods have been established, abundant information can be obtained in one analysis, and functional linking of analysis results is easy based on many research reports on single nucleotide polymorphisms and RNA functions. Nucleic acids derived from living organisms can be extracted from body fluids such as blood, secretions, tissues, etc., but it has recently been reported that RNA contained in skin surface lipids (SSL) can be used as a sample for analyzing living organisms, and that marker genes of the epidermis, sweat glands, hair follicles, and sebaceous glands can be detected from SSL (Patent Document 1). It has also 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] JP 2020-74769 A [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] Saeki et al., Journal of the Japanese Society of Dermatology (2021) 131: 2691-2777. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention relates to providing a method for measuring a gene or its expression product in order to detect the severity of atopic dermatitis. [Means for solving the problem]

[0008] The inventors collected SSLs from patients with atopic dermatitis of different severity and from healthy individuals, and comprehensively analyzed the expression state of RNA contained in the SSL as sequence information. As a result, they found that the expression levels of specific genes differed significantly between patients with different severity and between patients with mild to moderate disease and healthy individuals, and that this can be used as an indicator to detect the severity of atopic dermatitis. It has been known that there are genes whose expression varies between healthy people and atopic dermatitis patients, but it is not known whether all of these genes vary in expression depending on severity, such as blood TARC in mild to moderate patients compared to healthy people, and in severe patients compared to mild to moderate patients.In addition, even if the expression of a gene changes in mild to moderate patients or severe patients compared to healthy people, it does not necessarily vary in severe patients compared to mild to moderate patients, that is, depending on severity.Therefore, it is completely unexpected that the specific gene according to the present invention can be used as an indicator to detect different severity levels of atopic dermatitis.

[0009] That is, the present invention relates to the following 1) and 2). 1) A method for measuring a gene or its expression product in a subject, the method comprising a step of measuring the expression level of at least one gene or its expression product selected from a group of 16 genes consisting of C1QB, DIAPH1, LCE1A, CTSV, CTSZ, KRT72, SPINK5, KRT23, KLK5, LCE1B, SPRR1A, PKP1, KRT74, APRT, KRT25 and S100B in a biological sample collected from the subject in order to detect the severity of atopic dermatitis in 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 a nucleic acid derived therefrom, or an antibody that recognizes the expression product of the gene. Effect of the Invention

[0010] According to the present invention, it is possible to accurately and objectively detect the severity of atopic dermatitis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0012] In the present invention, the term "nucleic acid" or "polynucleotide" refers to DNA or RNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and "RNA" includes total RNA, mRNA, rRNA, tRNA, non-coding RNA, and synthetic RNA.

[0013] In the present invention, the term "gene" refers to 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 that contains some biological information 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 that code for their homologues (i.e., homologs or orthologs), mutants such as gene polymorphisms, and derivatives.

[0014] In the present invention, the term "expression product" of a gene is a concept that encompasses the transcription product and translation product of the gene. A "transcription product" is an 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.

[0015] In the present invention, "atopic dermatitis" refers to a disease whose main pathogenic cause is itch-causing 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) family history or medical history (any or multiple diseases among bronchial asthma, allergic rhinitis / conjunctivitis, and atopic dermatitis), or ii) a predisposition to easily produce IgE antibodies.

[0016] 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 skin rash, for example, as no symptoms (healthy), slight, mild (mild), moderate (moderate), or severe (severe). The characteristics of the rash include erythema, edema / infiltration / papule (solid, serous), exudate / crust, and excoriation. These include scars, lichenification, dryness, pruritus, prurigo nodules, scales (pityriasis, foliacea, membranous, etc.), blisters, pustules, erosions, and ulcers. The severity is classified using the Eczema Area and Severity Index (EASI), for example.<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 (JDJ 2001; 111: 2023-2033, JDJ 1998; 108: 1491-1496.), Severity Scoring of Atopic Dermatitis (SCORAD)<Dermatology, 1993; 186: 23-31.> etc. are known.

[0017] For example, the EASI is a value of 0 to 72 calculated based on the four symptom scores of erythema, edema / infiltration / papule, scratch, and lichenification at each evaluation site, and the percentage (%) of the area of ​​the top four symptoms in the entire evaluation site, with the head and neck, trunk, upper limbs, and lower limbs as evaluation sites. The head and neck EASI is calculated by multiplying the value calculated based on the four symptom scores of erythema, edema / infiltration / papule, scratch, and lichenification at the head and neck and the percentage (%) of the area of ​​the top four symptoms in the head and neck by 0.1, and is a value of 0 to 7.2. The severity evaluation based on the head and neck EASI is also used as one of the guidelines for administration of atopic dermatitis treatment drugs. In the present invention, it is preferable to use the severity classification based on the Head and Neck EASI to determine the severity of atopic dermatitis.

[0018] In the present invention, the severity of atopic dermatitis can be detected as "no symptoms", "mild to moderate symptoms" and "severe symptoms". However, it is preferable to distinguish between "no symptoms" and "mild to moderate symptoms", and between "mild to moderate symptoms" and "severe symptoms". In the present invention, "mild to moderate" includes mild and moderate symptoms (mild to moderate), for example, a head and neck EASI score of more than 0 and less than 2.4. "Severe" refers to severe, for example, a head and neck EASI score of 2.4 or more and 7.2 or less. "No symptoms" refers to a healthy person from the beginning, or a person in remission or near remission with no symptoms and at the same level as a healthy person. In addition, when assessing "no symptoms," "mild / moderate," and "severe" symptoms using the EASI score, the range of scores used as the basis for each assessment is not limited to the above and can be determined as appropriate.

[0019] 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 this specification, the terms "detection," "examination," "measurement," "judgment," or "evaluation" do not include a diagnosis of atopic dermatitis by a doctor.

[0020] In the present invention, the gene to be measured (hereinafter also referred to as "target gene") is at least one selected from a total of 16 genes, including 11 genes shown in Table 1 and 5 genes shown in Table 2. The gene names (Gene Symbols) and Gene IDs shown in Tables 1 and 2 are the Official Sites listed in NCBI ([www.ncbi.nlm.nih.gov / ]). The target gene or its expression product is a marker for detecting the severity of atopic dermatitis in the present invention, and the genes or their expression products shown in Table 1 are collectively referred to as the markers of Table 1, and the genes or their expression products shown in Table 2 are collectively referred to as the markers of Table 2. The marker of the present invention may be a gene or its expression product shown in Table 1 or Table 2 below, or a combination thereof. In one embodiment, the marker of the present invention is a nucleic acid marker such as DNA of the gene or RNA which is a transcription product thereof. In another embodiment, the marker of the present invention is a protein marker which is a translation product of the gene. Preferably, the marker of the present invention is a nucleic acid marker, more preferably an RNA marker.

[0021] [Table 1]

[0022] [Table 2]

[0023] The genes shown in Tables 1 and 2 include those consisting of nucleotide sequences registered with NCBI, as well as those consisting of sequences substantially identical to the registered sequences, so long as the genes themselves or expression products derived therefrom function as markers for detecting the severity of atopic dermatitis. Here, substantially identical sequences refer to sequences that have 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 gene 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.

[0024] As shown in the Examples below, the 11 genes shown in Table 1 are negative markers whose expression levels decrease stepwise with the severity of atopic dermatitis. Specifically, the genes in Table 1 are genes related to epidermal function selected from genes whose expression decreases in mild to moderate patients compared to healthy subjects and in severe patients compared to mild to moderate patients, and have a higher accuracy in determining severity than CCL17 (Gene ID: 6361), a gene encoding the TARC protein already known to reflect the severity of atopic dermatitis. On the other hand, the five genes shown in Table 2 are positive markers whose expression levels increase stepwise with the severity of atopic dermatitis. Specifically, the genes in Table 2 are immune function-related genes selected from genes whose expression increases in mild to moderate patients compared to healthy subjects and in severe patients compared to mild to moderate patients, and have a higher accuracy in determining severity than CCL17, a gene encoding the TARC protein already known to reflect the severity of atopic dermatitis. Therefore, the markers in Tables 1 and 2 make it possible to distinguish between healthy subjects and subjects with mild to moderate atopic dermatitis, and between subjects with mild to moderate atopic dermatitis and subjects with severe atopic dermatitis. Either the former negative marker or the latter positive marker may be used, or both may be used in combination.

[0025] The method of the present invention for measuring a gene or its expression product in a subject to detect the severity of atopic dermatitis in the subject comprises a step of measuring the expression level of at least one gene selected from the above-mentioned target genes or its expression product in a biological sample collected from the subject.

[0026] The subject for detecting the severity of atopic dermatitis in the subject of the present invention is not particularly limited in terms of sex, age, race, etc., and may include infants to the elderly. Preferably, the subject is a human who needs or desires to detect the severity of atopic dermatitis. 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.

[0027] The biological sample used in the present invention may be any cell, tissue, or biological material in which the expression of the target gene of the present invention changes depending on the severity of atopic dermatitis.Specific examples of the biological sample include organs, skin, blood, urine, saliva, sweat, stratum corneum, skin surface lipids (SSL), body fluids such as tissue exudates, serum and plasma prepared from blood, as well as feces, hair, etc., and preferably includes skin or skin surface lipids (SSL), and more preferably includes skin surface lipids (SSL).

[0028] Here, "skin surface lipids (SSL)" refers to the fat-soluble fraction present on the surface of the skin, and is sometimes called sebum. In general, SSL mainly contains secretions secreted from exocrine glands such as sebaceous glands in the skin, and is present on the skin surface in the form of a thin layer covering the skin surface. SSL contains RNA expressed in skin cells (see Patent Document 1). In the present invention, "skin" is a general term for an area including tissues such as the epidermis, dermis, hair follicles, sweat glands, sebaceous glands, and other glands on the body surface, unless otherwise specified.

[0029] Any means used for recovering or removing SSL from the skin can be used to collect SSL from the skin of a subject. Preferably, an SSL absorbent material, an SSL adhesive material, or an instrument for scraping SSL from the skin, as described below, can be used. The SSL absorbent material or SSL adhesive material is not particularly limited as long as it has an affinity for SSL, and examples thereof include polypropylene, pulp, and the like. More detailed examples of procedures for collecting SSL from the skin include a method of absorbing SSL into a sheet-like material such as oil blotting paper or oil blotting film, a method of adhering SSL to a glass plate, tape, or the like, and a method of scraping SSL off and collecting it with a spatula, scraper, or the like. In order to improve the adsorption of SSL, an SSL absorbent material that has been previously impregnated with a highly lipid-soluble solvent may be used. On the other hand, if the SSL absorbent material contains a highly water-soluble solvent or moisture, the adsorption of SSL is inhibited, so it is preferable that the content of highly water-soluble solvent or moisture is low. It is preferable to use the SSL absorbent material in a dry state. The site of the skin from which the SSL is collected is not particularly limited, and may be any site of the body such as the head, face, neck, trunk, hands and feet, with sites where sebum is secreted in large amounts, such as the skin of the face, being preferred. The site of the skin from which the SSL is collected may be either a rash site where atopic dermatitis has developed or a non-rash site where atopic dermatitis has not developed, but is preferably a rash site or a non-rash site adjacent to the rash site. Here, the vicinity of the rash site refers to a range within 10 cm adjacent to the rash site.

[0030] The RNA-containing SSL collected from the subject may be stored for a certain period of time. In order to minimize the degradation of the RNA contained in the SSL, it is preferable to store the collected SSL under low temperature conditions as soon as possible after collection. The temperature condition for storing the RNA-containing SSL in the present invention may be 0° C. or lower, preferably -20° C.±20° C. to -80° C.±20° C., more preferably -20° C.±10° C. to -80° C.±10° C., even more preferably -20° C.±20° C. to -40° C.±20° C., even more preferably -20° C.±10° C. to -40° C.±10° C., even more preferably -20° C.±10° C., and even more preferably -20° C.±5° C. The period for storing the RNA-containing SSL under the low temperature conditions is not particularly limited, but is preferably 12 months or less, for example, 6 hours or more and 12 months or less, more preferably 6 months or less, for example, 1 day or more and 6 months or less, even more preferably 3 months or less, for example, 3 days or more and 3 months or less.

[0031] In the present invention, examples of objects for measuring the expression level of a target gene or its expression product include cDNA artificially synthesized from RNA, DNA encoding the RNA, a protein encoded by the RNA, a molecule that interacts with the protein, a molecule that interacts with the RNA, or a molecule that interacts with the DNA. Here, examples of molecules that interact with RNA, DNA, or proteins include DNA, RNA, proteins, polysaccharides, oligosaccharides, monosaccharides, lipids, fatty acids, and phosphorylations, alkylations, sugar adducts, etc., of these, and complexes of any of the above. Furthermore, the expression level comprehensively refers to the expression amount and activity of the gene or expression product.

[0032] In a preferred embodiment of the method of the present invention, an SSL is used as a biological sample, in which case the expression level of RNA contained in the SSL is analyzed; specifically, the RNA is converted to cDNA by reverse transcription, and then the cDNA or its amplification product is measured. RNA extraction from SSL can be performed using methods commonly used for RNA extraction or purification from biological samples, such as the phenol / chloroform method, AGPC (acid guanidinium chloride) method, etc. Methods that can be used include the tetrahydrofuran (TRIzol) thiocyanate-phenol-chloroform extraction method, methods using columns such as TRIzol (registered trademark), RNeasy (registered trademark), and 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.

[0033] For the reverse transcription, a primer targeting a specific RNA to be analyzed may be used, but for more comprehensive nucleic acid storage and analysis, it is preferable to use a random primer. For the reverse transcription, a general reverse transcriptase or reverse transcription reagent kit can be used. Preferably, a highly accurate and efficient reverse transcriptase or reverse transcription reagent kit is used, and examples thereof include M-MLV Reverse Transcriptase and its variants, or commercially available reverse transcriptase or reverse transcription reagent kits, such as PrimeScript (registered trademark) Reverse Transcriptase series (Takara Bio Inc.), SuperScript (registered trademark) Reverse Transcriptase series (Thermo Scientific Co., Ltd.), etc. SuperScript (registered trademark) III Reverse Transcriptase, SuperScript (registered trademark) VILO cDNA Synthesis kit (both Thermo Scientific Co., Ltd.), etc. 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.

[0034] 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 represented by real-time RT-PCR, multiplex PCR, SmartAmp, LAMP and the like, 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 combinations of these.

[0035] In PCR, a primer pair targeted to a specific DNA to be analyzed may be used to amplify only one 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 for simultaneously amplifying multiple gene regions 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 temperature of the annealing and extension reaction in the PCR depends on the primers used and cannot be generally stated, but when the above multiplex PCR kit is used, it is 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 reaction are preferably performed in one step. The time of the annealing and extension reaction steps can be adjusted depending on the size of the DNA to be amplified, etc., but is preferably 14 to 18 minutes. The conditions of 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 above-mentioned temperature and time can be performed using a thermal cycler generally used for PCR.

[0036] 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, for example, by a size separation column, a size separation chip, magnetic beads usable for size separation, etc. A preferred example of magnetic beads usable for size separation includes Solid Phase Reversible Immobilization (SPRI) magnetic beads such as Ampure XP.

[0037] The purified PCR reaction product may be subjected to further processing required 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, the PCR primer sequence may be removed from the amplified DNA, and adapter ligation may be performed, and the resulting reaction product may be amplified as necessary to prepare a library for quantitative analysis. These operations may be performed, for example, using the 5×VILO RT Reaction Mix included in the SuperScript (registered trademark) VILO cDNA Synthesis kit (Life Technologies Japan, Inc.), and the 5×Ion AmpliSeq HiFi Mix and Ion AmpliSeq Transcriptome Human Gene Expression Kit (Life Technologies Japan, Inc.) and the Ion AmpliSeq Transcriptome Human Gene Expression Core Panel included in the Ion AmpliSeq Transcriptome Human Gene Expression Kit according to the protocol included with each kit.

[0038] When measuring the expression level of a target gene or a nucleic acid derived therefrom using the Northern blot hybridization method, for example, first, a probe DNA is 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 conventional manner. Thereafter, the formed double strand of labeled DNA and RNA is measured by detecting a signal derived from the label.

[0039] 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 in a conventional manner, and then a pair of primers (a positive strand that binds to the above 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 with this. Then, PCR is performed in a conventional manner, and the resulting amplified double-stranded DNA is detected. The amplified double-stranded DNA can be detected by a method of detecting labeled double-stranded DNA produced by performing the above PCR using primers that have been labeled in advance with RI, a fluorescent substance, or the like.

[0040] 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, the "partial sequence" may be a nucleic acid consisting of at least 15 to 25 bases. Here, the stringent conditions may be washing conditions of about "1xSSC, 0.1% SDS, 37°C", more stringent hybridization conditions may be about "0.5xSSC, 0.1% SDS, 42°C", and even more stringent hybridization conditions may be about "0.1xSSC, 0.1% SDS, 65°C". Hybridization conditions are described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001), etc.

[0041] 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.

[0042] The probe or primer used in the above measurement, i.e., a primer for specifically recognizing and amplifying the target gene of the present invention or a nucleic acid derived therefrom, or a probe for specifically detecting the RNA or a nucleic acid derived therefrom, corresponds to this, and these can be designed based on the base sequence constituting the target gene. Here, "specifically recognize" means that the detected matter or product can be determined to be the gene or a nucleic acid derived therefrom, for example, in the Northern blot method, so that substantially only the target gene of the present invention or a nucleic acid derived therefrom can be detected, and for example, in the RT-PCR method, so that substantially only the nucleic acid is amplified. Specifically, a DNA consisting of a 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, with respect to the other strand. In addition, "complementary" is not limited to a completely complementary sequence in the certain number of consecutive nucleotide regions, but may have a base sequence identity of preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more. The identity of the base sequence can be determined by an algorithm such as BLAST. When used as a primer, such an oligonucleotide may be capable of specific annealing and chain elongation, and may generally have 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, such an oligonucleotide may be capable of specific hybridization, and may have at least a part or all of the sequence of DNA (or its complementary strand) consisting of the base sequence constituting the target gene of the present invention, and may have 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. In this case, the "oligonucleotide" may be DNA or RNA, and may be either synthetic or natural. Furthermore, the probe used in hybridization is usually labeled.

[0043] In addition, 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. It 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 a biological sample with an antibody that specifically recognizes the expression product of the present invention, specifically an antibody that recognizes a structural characteristic site (epitope) that can distinguish the expression product protein from other proteins, detecting the polypeptide or protein in the sample that binds to the antibody, and measuring its level. For example, according to the Western blot method, after using the above-mentioned antibody as the primary antibody, the primary antibody is labeled with a radioisotope, a fluorescent substance, an enzyme, or the like as the secondary antibody, and the primary antibody is then labeled, and signals derived from these labeling substances are measured with a radiation measuring device, 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 by immunizing a non-human animal such as a rabbit with a protein expressed in E. coli or the like and purified according to a conventional method, or by synthesizing a partial polypeptide of the protein according to a conventional method, and then from the serum of the immunized animal according to a conventional method. 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 that has been expressed and purified in E. coli or a partial polypeptide of the protein according to a conventional method, and fusing the resulting spleen cells with myeloma cells. Monoclonal antibodies may also be prepared using phage display (Griffiths, AD; Duncan, AR, Current Opinion in Biotechnology, Volume 9, Number 1, February 1998, pp. 102-108(7)).

[0044] 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 may be performed 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, the read count value, which is the data on the expression level, the RPM value obtained by correcting the read count value for the difference in the total number of reads between samples, and the value obtained by converting the RPM value to a logarithmic value with base 2 (Log2 RPM value) or the logarithm of base 2 with an integer 1 added (Log2(RPM+1) value), or Normalized counts were calculated using DESeq2 (Love MI et al. Genome Biol. 2014) or logarithm of base 2 with an integer 1 added (Log2(c It is preferable to use the index of the number of copies (number of copies + 1) as the index. In addition, it may be a value calculated by fragments per kilobase of exon per million reads mapped (FPKM), reads per kilobase of exon per million reads mapped (RPKM), transcripts per million (TPM), etc., which are common quantitative values ​​for RNA-seq. It may also be a signal value obtained by a microarray method and its corrected value. In addition, when analyzing only a specific target gene by RT-PCR or the like, it is preferable to use a method in which the expression level of the target gene is converted to a relative expression level based on the expression level of a housekeeping gene as a reference for analysis, or a method in which the absolute copy number is quantified (absolute quantification) using a plasmid containing the region of the target gene for analysis. It may also be the copy number obtained by a digital PCR method. Here, the "control level" refers to, for example, the expression level of the target gene or its expression product in healthy individuals when detecting mild to moderate patients, and in mild to moderate patients when detecting severe patients. The expression levels of healthy individuals, mild to moderate patients, and severe patients may be statistical values ​​(e.g., average values, etc.) of the expression levels of the gene or its expression product measured from a group of healthy individuals, mild to moderate patients, and severe patients. When there are multiple target genes, it is preferable to determine the control level for each gene or its expression product.

[0045] In addition, the severity of atopic dermatitis in the present invention can also be detected by comparing the expression level of the target gene or its expression product of the present invention with the cutoff value (reference value) of each gene or its expression product. The cutoff value may be determined as appropriate based on statistical values ​​such as the average value and standard deviation of the expression levels based on the expression levels of the target gene or its expression product in healthy subjects, mildly ill patients, moderately ill patients, and severely ill patients, which are previously obtained as reference data.

[0046] The method of determining the cutoff value (reference value) is not particularly limited, and can be determined according to a known method. For example, it can be obtained from a ROC (Receiver Operating Characteristic Curve) curve. In the ROC curve, the vertical axis plots the probability of a positive result in a positive patient (sensitivity or true positive rate), 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). With regard to the "true positive rate (sensitivity)" and "false positive rate (1-specificity)" shown in the ROC curve, the threshold value at the point (Youden index) where "true positive rate (sensitivity)" - "false positive rate (1-specificity)" is maximum can be used as the cutoff value (reference value).

[0047] For example, a cutoff value (reference value) for distinguishing between two groups of different severity is determined in advance for the genes shown in Table 1, and if the expression level of a target gene or its expression product in a biological sample derived from a subject is equal to or higher than the cutoff value (reference value) for each gene or its expression product, the group is determined to be less severe, whereas if the expression level is equal to or lower than the reference value, the group is determined to be more severe.

[0048] In addition, for example, a cutoff value (reference value) for distinguishing between two groups of different severity is determined in advance for the genes shown in Table 2, and if the expression level of a target gene or its expression product in a biological sample derived from a subject is below the cutoff value (reference value) of each gene or its expression product, the group is determined to be less severe, and if the expression level is above the reference value, the group is determined to be more severe.

[0049] As shown in Table 3 below, when the 16 target genes of the present invention were used to discriminate between two groups, healthy subjects and subjects with mild to moderate atopic dermatitis, and between subjects with mild to moderate atopic dermatitis and subjects with severe atopic dermatitis, the average values ​​(AUC3) of the areas under the curve (AUC1 and AUC2) of the ROC curves for discriminating between the two groups were all higher than the AUC3 obtained when the CCL17 gene, which encodes the TARC protein known as a severity marker, was used, demonstrating high discrimination accuracy. As described above, the cutoff value (reference value) can be determined by the threshold value at the point (Youden index) where the "true positive rate (sensitivity)" - "false positive rate (1 - specificity)" is maximum for the "true positive rate (sensitivity)" and "false positive rate (1 - specificity)" shown in the ROC curve. Table 3 shows an example of each reference value when discriminating between two groups, healthy and mild to moderate atopic dermatitis, and between two groups, mild to moderate atopic dermatitis and severe atopic dermatitis, using 16 genes, but the reference values ​​are not limited to these and can be determined appropriately.

[0050] In another embodiment, the expression level of a target gene or its expression product in a subject is measured, and the measured expression level is compared with a preset reference value, thereby detecting the severity of atopic dermatitis in the subject. The reference value can be determined in advance based on the relationship between the severity of atopic dermatitis (the severity level of atopic dermatitis classified based on the score value related to the severity of atopic dermatitis or the score value related to the severity of atopic dermatitis) and the expression level of the target gene or its expression product. For example, a certain population can be divided into a plurality of groups of different severity based on the severity of atopic dermatitis, and a reference value for determining whether or not the subject belongs to each group can be determined with reference to the statistical value (e.g., average value) of the expression level of the target gene or its expression product in each group. When a plurality of types of markers are used as the target gene or its expression product, it is preferable to determine the reference value for each marker. The population may be any of a group of patients with atopic dermatitis, a group of healthy subjects and a group of patients with atopic dermatitis, and a group of patients with a specific severity of atopic dermatitis. In addition, groups may be created by age, generation, sex, or race according to the subjects to be detected. Examples of groups used for calculating the reference value include a mild patient group (mild group), a moderate patient group (moderate group), a mild and moderate patient group (mild-moderate group), a severe patient group (severe group), etc. A healthy control group (a group without symptoms of atopic dermatitis) may also be included.

[0051] When the negative marker is used, the lower the expression level, the higher the severity of the subject's atopic dermatitis is detected, whereas when the positive marker is used, the higher the expression level, the higher the severity of the subject's atopic dermatitis is detected.

[0052] The specific method for setting the reference value and for classifying the severity based on the reference value can be appropriately carried out according to the common technical knowledge of those skilled in the art.

[0053] In yet another embodiment, the expression level of a target gene or its expression product in a subject is measured at different times, and the measured expression levels of the target gene or its expression product are compared to detect a change in the severity (e.g., exacerbation or amelioration) of the subject's atopic dermatitis.

[0054] When a negative marker is used, an increase in its expression level over time represents a decrease in the severity of the subject's atopic dermatitis, while a decrease in its expression level over time represents an increase in the severity of the subject's atopic dermatitis. In one example, the expression level of the target gene or its expression product in the same subject in a past measurement is taken as a reference value. When the expression level of the negative marker measured from the subject is higher than the reference value, the severity of the subject's atopic dermatitis is detected as decreased, while when the expression level of the negative marker measured from the subject is lower than the reference value, the severity of the subject's atopic dermatitis is detected as increased. If necessary, the severity of the subject's atopic dermatitis may be determined by a conventional method during the past measurement. In this case, when the expression level of the negative marker measured from the subject is higher or lower than the reference value, the severity of the subject's atopic dermatitis can be detected as lower or higher than the severity of the atopic dermatitis in the past measurement.

[0055] When a positive marker is used, an increase in its expression level over time represents an increase in the severity of the subject's atopic dermatitis, while a decrease in its expression level over time represents a decrease in the severity of the subject's atopic dermatitis. In one example, the expression level of the target gene or its expression product in the same subject in a past measurement is taken as a reference value. When the expression level of the positive marker measured from the subject is higher than the reference value, the severity of the subject's atopic dermatitis is detected as having increased, while when the expression level of the positive marker measured from the subject is lower than the reference value, the severity of the subject's atopic dermatitis is detected as having decreased. If necessary, the severity of the subject's atopic dermatitis may be determined by a conventional method during the past measurement. In this case, when the expression level of the positive marker measured from the subject is higher or lower than the reference value, the severity of the subject's atopic dermatitis can be detected as being higher or lower than the severity of the atopic dermatitis in the past measurement.

[0056] In one embodiment of the method of the present invention, if the expression level of the target gene or its expression product of the present invention derived from a subject is preferably 91% or less, more preferably 83% or less, and even more preferably 77% or less of the standard value, the expression level of the target gene or its expression product can be determined to be lower than the standard value, and if the expression level of the target gene or its expression product of the present invention is preferably 110% or more, more preferably 120% or more, and even more preferably 130% or more of the standard value, the expression level of the target gene or its expression product can be determined to be higher than the standard value. Alternatively, the difference between the expression level of the target gene or its expression product derived from a subject and the standard value can be determined, for example, by whether or not the two are statistically significantly different. When multiple markers are used as the target gene or its expression product, the expression level of each target gene or its expression product is compared with the standard value, and the severity of atopic dermatitis can be detected by examining whether or not a certain proportion, for example, 50% or more, preferably 70% or more, more preferably 90% or more, and even more preferably 100% of the expression levels of the target genes or their expression products are different from the standard value.

[0057] In another embodiment, the expression level of a target gene or its expression product in a subject is measured, and the expression level is substituted into a discriminant (prediction model) for detecting the severity of atopic dermatitis, thereby detecting the severity of atopic dermatitis in the subject. Specifically, a discriminant can be constructed by machine learning using measured values ​​of the expression levels of target genes or their expression products from patients with atopic dermatitis of different severity (e.g., mild patients, mild-moderate patients, moderate patients, severe patients) and healthy individuals as teacher samples, the expression levels of at least one or more target genes or their expression products obtained from each person in the teacher samples as explanatory variables, and the severity of atopic dermatitis of each person (e.g., mild, mild-moderate, moderate, severe) or no symptoms (healthy) as the objective variable. The expression levels of the target genes or their expression products in the subject are then measured, and the severity of atopic dermatitis in the subject can be detected by substituting the expression levels into the discriminant. In creating a discriminant equation, dimensionality can be reduced by principal component analysis (PCA) and the main components can be used as explanatory variables.

[0058] As an algorithm for constructing the discriminant, known algorithms such as algorithms used in machine learning can be used. Examples of machine learning algorithms include random forest, support vector machine with linear kernel (SVM linear), support vector machine with rbf kernel (SVM rbf), neural net, generalized linear model, regularized linear discriminant analysis, regularized logistic regression, etc. A predicted value is calculated by inputting data for verification into the constructed prediction model, and a model whose predicted value is most compatible with the actual measured value, for example, a model with the highest accuracy rate, can be selected as the optimal prediction model. In addition, the detection rate (Recall), precision, and the F value, which is the harmonic mean thereof, can be calculated from the predicted value and the actual measured value, and the model with the highest F value can be selected as the optimal prediction model.

[0059] The test kit for detecting the severity of atopic dermatitis of the present invention contains a test reagent for measuring the expression level of the target gene of the present invention or its expression product in a biological sample separated from a patient. Specifically, the test kit includes a reagent for nucleic acid amplification or hybridization containing an oligonucleotide (e.g., a primer for PCR) that specifically binds (hybridizes) to the target gene of the present invention or a nucleic acid derived therefrom, or a reagent for immunological measurement containing an antibody that recognizes the expression product (protein) of the target gene of the present invention. The oligonucleotide, antibody, etc. contained in the kit can be obtained by a known method 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).

[0060] In relation to the above-described embodiment, the present invention further discloses the following aspects. <1> A method for measuring a gene or its expression product in a subject, the method comprising a step of measuring the expression level of at least one gene or its expression product selected from a group of 16 genes consisting of C1QB, DIAPH1, LCE1A, CTSV, CTSZ, KRT72, SPINK5, KRT23, KLK5, LCE1B, SPRR1A, PKP1, KRT74, APRT, KRT25 and S100B in a biological sample collected from the subject in order to detect the severity of atopic dermatitis in the subject. <2> 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 16 genes consisting of C1QB, DIAPH1, LCE1A, CTSV, CTSZ, KRT72, SPINK5, KRT23, KLK5, LCE1B, SPRR1A, PKP1, KRT74, APRT, KRT25 and S100B in a biological sample collected from the subject. <3> The gene is at least one gene selected from the group of nine genes consisting of C1QB, DIAPH1, LCE1A, CTSV, CTSZ, KRT72, SPINK5, KRT23 and KLK5, preferably C1QB; <1> or <2> The method according to <4> the gene is at least one gene selected from a group of 11 genes consisting of LCE1A, CTSV, KRT72, SPINK5, KRT23, KLK5, LCE1B, SPRR1A, PKP1, KRT74, and KRT25, preferably a group of 6 genes consisting of LCE1A, CTSV, KRT72, SPINK5, KRT23, and KLK5, and the lower the expression level of the gene or its expression product, the higher the severity of the atopic dermatitis in the subject is detected. <1> or <2> How to. <5> A reference value for discriminating between two groups with different severity is determined in advance, and if the expression level of the gene or its expression product of the atopic dermatitis patient is equal to or higher than the reference value, the patient is classified as a group with a lower severity, whereas if the expression level is equal to or lower than the reference value, the patient is classified as a group with a higher severity. <4> How to. <6> The gene is at least one gene selected from a group of five genes consisting of C1QB, DIAPH1, CTSZ, APRT, and S100B, preferably a group of three genes consisting of C1QB, DIAPH1, and CTSZ, and the higher the expression level of the gene or its expression product, the higher the severity of the atopic dermatitis in the subject is detected. <1> or <2> How to. <7> A reference value for discriminating between two groups with different severity levels is determined in advance, and if the expression level of the gene or its expression product of the atopic dermatitis patient is equal to or lower than the reference value, the patient is classified as a group with a lower severity level, and if the expression level is equal to or higher than the reference value, the patient is classified as a group with a higher severity level. <6> How to. <8> The severity is one or more selected from no symptoms (healthy), mild, moderate, and severe, <1> ~ <7> Either way. <9> The two groups with different severity are normal and mild to moderate atopic dermatitis, or mild to moderate atopic dermatitis and severe atopic dermatitis. <5> or <7> How to. <10> A discriminant equation is constructed using measured values ​​of the expression levels of the target gene or its expression product from a subject, the measured values ​​being derived from patients with atopic dermatitis having different severity levels and healthy subjects, as teacher samples, and the measured values ​​of the expression levels of the target gene or its expression product from the subject are substituted into the discriminant equation to detect the severity of atopic dermatitis in the subject. <1> or <2> How to. <11> The severity is one or more selected from mild, mild-moderate, moderate and severe. <10> How to. <12> The discriminant equation is constructed by machine learning using the expression levels of at least one or more target genes or their expression products obtained from each person in the teacher sample as explanatory variables, and the severity of atopic dermatitis and the absence of symptoms (healthy state) of each person as objective variables. <10> or <11> How to. <13> The algorithm used for machine learning is selected from the group consisting of random forest, support vector machine with linear kernel (SVM linear), support vector machine with rbf kernel (SVM rbf), neural net, generalized linear model, regularized linear discriminant analysis, and regularized logistic regression. <12> How to. <14> The expression level of the gene or its expression product is the expression level of mRNA. <1> ~ <13> Either way. <15> The gene or its expression product is RNA contained in the lipids on the skin surface of the subject. <1> ~ <14> Either way. <16> The severity is classified based on the Head and Neck EASI. <1> ~ <15> Either way. <17> 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> ~ <16> A test kit for detecting the severity of atopic dermatitis, which is used in any one of the methods above. <18> Use of at least one gene or its expression product selected from a group of 16 genes consisting of C1QB, DIAPH1, LCE1A, CTSV, CTSZ, KRT72, SPINK5, KRT23, KLK5, LCE1B, SPRR1A, PKP1, KRT74, APRT, KRT25 and S100B derived from a biological sample collected from a subject as a marker for the severity of atopic dermatitis. <19> The gene or its expression product is at least one gene or its expression product selected from the group of nine genes consisting of C1QB, DIAPH1, LCE1A, CTSV, CTSZ, KRT72, SPINK5, KRT23, or KLK5, preferably C1QB or its expression product. <18> Use of. <20> The gene or its expression product is mRNA derived from skin surface lipids collected from the subject. <18> or <19> Use of. <21> The severity is based on the Head and Neck EASI. <18> ~ <20> Use of either. EXAMPLES

[0061] 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 and SSL collection of patients with mild to moderate atopic dermatitis The subjects were 14 healthy subjects (25-57 years old, male) and 29 adults with atopic skin (AD) (23-56 years old, male). The atopic dermatitis patients had been diagnosed by a dermatologist with mild to moderate severity of atopic dermatitis. The head and neck EASI score (Hanifin et al. Exp dermatol.10, 2001) was used for diagnosis, and the head and neck EASI score of the patients was 0.1 to 1.8 (average 0.50). Sebum was collected from the entire face of each subject using a sheet of oil blotting film (polypropylene, 5.0 cm x 8.0 cm, 3M Co.), and the oil blotting film was transferred to a vial and stored at -80 °C for about one month until it was used for RNA extraction.

[0062] 2) Diagnosis of patients with severe atopic dermatitis and SSL sampling Thirty healthy subjects (24-59 years old, male) and 30 adults with atopic skin (AD) (21-59 years old, male) were used as subjects. The atopic dermatitis patients were diagnosed by a dermatologist as having severe atopic dermatitis. The head and neck EASI score (Hanifin et al. Exp dermatol.10, 2001) was used for diagnosis, and the head and neck EASI score of the patients was 2.4-6.6 (average 3.0). Sebum was collected from the entire face of each subject using one oil blotting film (polypropylene, 5.0 cm x 8.0 cm, 3M Co.), and the oil blotting film was transferred to a vial and stored at -80 ° C for about one month until it was used for RNA extraction.

[0063] 3) RNA preparation and sequencing The oil blotting film from which sebum was collected in 1) and 2) above was cut to an appropriate size, and RNA was transferred to the aqueous layer using QIAzol Lysis Reagent (Qiagen) according to the attached protocol. RNA was extracted from the aqueous layer using a commercially available RNA extraction kit using a spin column for RNA extraction according to the attached protocol. The extracted RNA was reverse transcribed at 42°C for 90 minutes using SuperScript VILO cDNA Synthesis kit (Life Technologies Japan, Inc.) to synthesize cDNA. The random primers included in the kit were used as primers for the reverse transcription reaction. A library containing DNA derived from the 20802 gene was prepared from the obtained cDNA by multiplex PCR. Multiplex PCR was performed using Ion AmpliSeq Transcriptome Human Gene Expression Kit (Life Technologies Japan, Inc.) under the conditions of [99°C, 2 minutes → (99°C, 15 seconds → 62°C, 16 minutes) × 20 cycles → 4°C, Hold]. The PCR products obtained were purified with Ampure XP (Beckman Coulter, Inc.), followed by buffer reconstitution, digestion of primer sequences, adapter ligation and purification, and amplification to prepare libraries. The prepared libraries were loaded onto an Ion 540 Chip and sequenced using an Ion S5 / XL system (Life Technologies Japan, Inc.). Each read sequence obtained by sequencing was genetically mapped using the hg19 AmpliSeq Transcriptome ERCC v1, a reference sequence for the human genome, to determine the gene from which each read sequence originated.

[0064] 4) Data analysis: Genes whose expression changes with severity according to the head and neck EASI score i) Usage Data The expression data (read count values) of SSL-derived RNA from a total of 44 healthy subjects, 29 mild to moderate AD patients, and 30 severe AD patients measured in 3) above were corrected using a method called DESeq2. However, only 7177 genes for which expression data that was not a missing value was obtained in 90% or more of the expression data of all sample subjects were used in the following analysis. The count values ​​corrected using a method called DESeq2 (Normalized count values) were used for the analysis.

[0065] ii) RNA expression analysis 1 Based on the SSL-derived RNA expression levels (normalized count values) of healthy subjects and mild to moderate AD subjects measured in i) above, RNAs (genes with altered expression) with a p-value corrected value (FDR) of less than 0.1 in mild to moderate AD compared to healthy subjects were identified. As a result, 1424 types of RNA were decreased (DOWN) in mild to moderate AD, and 1966 types of RNA were increased (UP) in mild to moderate AD.

[0066] iii) RNA expression analysis 2 Based on the SSL-derived RNA expression levels (normalized count values) of mild-moderate AD and severe AD measured in i) above, RNAs (genes with altered expression) with a p-value corrected value (FDR) of less than 0.1 in severe AD compared to mild-moderate AD were identified. As a result, 1436 types of RNA were decreased (DOWN) in severe AD, and 1417 types of RNA were increased (UP) in severe AD.

[0067] iv) Extraction of genes whose expression decreases stepwise with disease severity Genes that corresponded to both the 1424 genes whose expression was decreased in mild to moderate AD compared to healthy subjects identified in ii) above and the 1436 genes whose expression was decreased in severe AD compared to mild to moderate AD identified in iii) above were extracted, and 74 genes were found to be relevant. These 74 genes are genes whose expression may be gradually decreased according to the severity of AD, that is, genes whose expression level can detect the severity of atopic dermatitis. Among these genes, the relevant Reactome Pathways and GO terms were identified. Based on the genes that compose the epidermis and information on genes reported in literature that would be known to those skilled in the art, 13 genes were extracted as genes related to epidermal functions. These 13 genes were then used as candidates to select genes with higher discrimination accuracy in the evaluation in the following step 5).

[0068] v) Extraction of genes whose expression increases stepwise with disease severity Genes corresponding to both the 1966 genes whose expression was increased in mild to moderate AD compared to healthy subjects as identified in ii) above and the 1417 genes whose expression was increased in severe AD compared to mild to moderate AD as identified in iii) above were extracted, and 34 genes were found to be relevant. These 34 genes are genes whose expression may increase stepwise with the severity of AD, that is, genes whose expression level can detect the severity of atopic dermatitis. Among these genes, related reactome pathways and GO terms were identified. Based on the genes that compose the disease and information on genes reported in literature that would be known to those skilled in the art, nine genes were extracted as genes related to immune function. Among them was CCL17, a gene that codes for the TARC protein, which is already known to reflect the severity of AD. These nine genes were then used as candidates to select genes with higher discrimination accuracy in the evaluation in the following step 5).

[0069] 5) Selection of more accurate markers for determining severity i) Distinguishing between healthy subjects and mild to moderate AD The accuracy of discriminating between healthy and mild to moderate AD was evaluated using the ROC curve for the total of 22 genes extracted in iv) and v) of 4) above. When discriminating whether a sample is negative or positive using the magnitude of a certain continuous value as an index, the ROC curve plots the probability of a positive result in the positive group (sensitivity or true positive rate) on the vertical axis and the value obtained by subtracting the probability of a negative result in the negative group (specificity) from 1 (false positive rate) on the horizontal axis for the negative and positive groups. The larger the area under the curve (AUC) of the ROC curve, the higher the discrimination accuracy of the two groups is judged to be. In the evaluation of the accuracy of discriminating between healthy and mild to moderate AD using the 13 genes in iv) above, mild to moderate AD was set as the negative group, and healthy was set as the positive group. In the evaluation of the accuracy of discriminating between healthy and mild to moderate AD using the 9 genes in v) above, healthy was set as the negative group, and mild to moderate AD was set as the positive group. Then, the accuracy rate (accuracy rate 1) in the Youden index, the AUC value (AUC1) and the cutoff value (reference value 1) for each of the 22 genes were calculated.

[0070] ii) Distinguishing between mild and moderate AD The accuracy of discriminating between mild and moderate AD and severe AD was evaluated using ROC curves for the 22 genes extracted in iv) and v) above. In the evaluation of the accuracy of discriminating between mild and moderate AD and severe AD using the 13 genes in iv) above, severe AD was classified as the negative group and mild and moderate AD as the positive group. In the evaluation of the accuracy of discriminating between mild and moderate AD and severe AD using the 9 genes in v) above, mild and moderate AD was classified as the negative group and severe AD as the positive group. Then, the accuracy rate (accuracy rate 2) in the Youden index, the AUC value (AUC2) and the cutoff value (reference value 2) of each of the 22 genes were calculated.

[0071] iii) Selection of genes with higher accuracy suitable for discriminating between healthy, mild-moderate AD, and severe AD Among the 22 types of genes extracted in 4) above, the average value of AUC1 and AUC2 (AUC3) was calculated and used as an index to evaluate whether any gene can discriminate between healthy and mild-moderate AD and between mild-moderate AD and severe AD with high accuracy (Table 3). Then, AUC3=0.693 of CCL17, a gene encoding the TARC protein already known to reflect the severity of AD, was used as a comparison target, and genes with a higher AUC3 than this were extracted. As a result, 11 genes, LCE1A, CTSV, KRT72, SPINK5, KRT23, KLK5, LCE1B, SPRR1A, PKP1, KRT74, and KRT25, were extracted from the 13 types of genes extracted in iv) above in 4). Furthermore, five genes, C1QB, DIAPH1, CTSZ, APRT, and S100B, were extracted from the nine genes extracted in 4) v) above. The AUC1, accuracy rate 1, reference value 1, AUC2, accuracy rate 2, reference value 2, and AUC3 of the total of 16 extracted genes and the comparative subject CCL17 are shown in Table 3. In the table, "decrease" in the expression change indicates that expression decreases with worsening of severity, and "increase" indicates that expression increases with worsening of severity. From the above results, the 16 genes consisting of C1QB, DIAPH1, LCE1A, CTSV, CTSZ, KRT72, SPINK5, KRT23, KLK5, LCE1B, SPRR1A, PKP1, KRT74, APRT, KRT25 and S100B have a higher AUC3 than that of CCL17, i.e., they are considered to be genes that can distinguish between healthy subjects and mild-moderate AD, and between mild-moderate AD and severe AD with high accuracy.

[0072] [Table 3]

Claims

1. A method for measuring the expression level of mRNA of a gene in a subject, the method comprising the step of measuring the expression level of mRNA of at least one gene selected from a group of 16 genes consisting of KLK5, C1QB, DIAPH1, LCE1A, CTSV, CTSZ, KRT72, SPINK5, KRT23, LCE1B, SPRR1A, PKP1, KRT74, APRT, KRT25 and S100B, for lipids on the surface of the skin collected from the entire face of the subject in order to detect the severity of atopic dermatitis in the subject.

2. The method according to claim 1, wherein the mRNA of the gene is the mRNA of at least one gene selected from a group of nine genes consisting of C1QB, DIAPH1, LCE1A, CTSV, CTSZ, KRT72, SPINK5, KRT23 and KLK5.

3. The method according to claim 1, wherein the mRNA of the gene is the mRNA of at least one gene selected from a group of 11 genes consisting of LCE1A, CTSV, KRT72, SPINK5, KRT23, KLK5, LCE1B, SPRR1A, PKP1, KRT74 and KRT25, and the lower the expression level of the mRNA of the gene, the higher the severity of the atopic dermatitis of the subject is detected.

4. The method according to claim 3, wherein a reference value for discriminating between two groups of different severity is determined in advance, and if the expression level of mRNA of the gene of an atopic dermatitis patient is equal to or higher than the reference value, the patient is classified as a group of less severe severity, and if the expression level of mRNA of the gene of an atopic dermatitis patient is equal to or lower than the reference value, the patient is classified as a group of more severe severity.

5. The method according to claim 1, wherein the mRNA of the gene is the mRNA of at least one gene selected from a group of five genes consisting of C1QB, DIAPH1, CTSZ, APRT and S100B, and the higher the expression level of the mRNA of the gene, the higher the severity of the atopic dermatitis of the subject is detected.

6. The method according to claim 5, wherein a reference value for discriminating between two groups with different severity is determined in advance, and if the expression level of mRNA of the gene of atopic dermatitis patients is below the reference value, the group is discriminated as being less severe, and if the expression level of mRNA of the gene of atopic dermatitis patients is above the reference value, the group is discriminated as being more severe.

7. The method according to any one of claims 1 to 6, wherein the severity is one or more selected from the group consisting of no symptoms, mild to moderate symptoms, and severe symptoms.

8. The method according to claim 1, wherein the severity of atopic dermatitis in a subject is detected by substituting the measured value of the expression level of the mRNA of the target gene in a subject into a discriminant constructed using the measured values ​​of the expression level of the mRNA of the target gene from patients with atopic dermatitis having different severity and from healthy individuals as teacher samples.