Method for detecting severity of atopic dermatitis

Abstract

To provide a method for measuring a gene or its expression product for detecting the severity of atopic dermatitis.SOLUTION: A method for measuring a gene or an expression product thereof in a subject, comprising a step of measuring an expression level of at least one gene or an expression product thereof 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.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 thymus and activation-regulated chemokine (TARC) and squamous cell carcinoma antigen 2 (SCCA2) levels as indicators have been used to objectively assess the severity of atopic dermatitis. Because the levels of these substances in the blood increase with the severity of atopic dermatitis, they are used as indicators that sensitively reflect the progression of the disease, and are used not only to evaluate the onset and severity of atopic dermatitis, but also for patient education and determining 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] 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 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 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 patients with mild to moderate disease and healthy individuals, and that this could 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 individuals and atopic dermatitis patients, but it is not known whether all of these genes, like blood TARC, have expression changes depending on the severity, such as in mild to moderate patients compared to healthy individuals, 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 individuals, it does not necessarily mean that the expression changes in severe patients compared to mild to moderate patients, that is, depending on the severity.Therefore, it is completely unexpected that the specific gene of 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, 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. [Effects 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 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 "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" 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.

[0014] 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.

[0015] 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. Predisposition to atopy includes 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 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 rash, for example, as no symptoms (healthy), slight, mild (mild), moderate (moderate), or severe (severe). The characteristics of the rash include erythema, edema / infiltration / papules (solid, serous), exudate / crust, and excoriation. Symptoms include scars, lichenification, dryness, itching, prurigo nodules, scales (pityriasis, foliaceous, membranous, etc.), blisters, pustules, erosions, and ulcers. Severity classifications include, for example, 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.

[0017] For example, the EASI is a value ranging from 0 to 72 calculated based on the scores for four symptoms (erythema, edema / infiltration / papules, excoriation, and lichenification) at each evaluation site, and the percentage (%) of the area of ​​the entire evaluation site that is accounted for by these four symptoms. The head and neck EASI is calculated by multiplying the score for each of the four symptoms (erythema, edema / infiltration / papules, excoriation, and lichenification) at each evaluation site and the percentage (%) of the area of ​​the entire evaluation site that is accounted for by these four symptoms by 0.1, resulting in a value ranging from 0 to 7.2. The severity assessment based on the head and neck EASI is also used as a guide for the administration of atopic dermatitis therapeutic 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 detection of the severity of atopic dermatitis according to the present invention, "no symptoms," "mild to moderate," and "severe" can be detected, but it is preferable to distinguish between "no symptoms" and "mild to moderate," and between "mild to moderate" and "severe." In the present invention, "mild to moderate" includes mild and moderate symptoms (mild to moderate illness), and corresponds to, for example, a head and neck EASI score of greater than 0 and less than 2.4. "Severe" refers to severe illness, and corresponds to, for example, a head and neck EASI score of 2.4 or greater but less than 7.2. "No symptoms" refers to a person who was originally healthy, or to a person who is in remission or near remission and has no symptoms, at the same level as a healthy person. In addition, when assessing "no symptoms," "mild to moderate," and "severe" 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 also be expressed 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, consisting of 11 genes shown in Table 1 and 5 genes shown in Table 2. The gene names (Gene Symbols) and Genomes shown in Tables 1 and 2 are as follows: The ID is the Official S ID listed in NCBI ([www.ncbi.nlm.nih.gov / ]). The target genes or their expression products are referred to in accordance with the symbol and Gene ID. In the present invention, the target genes or their expression products can be said to be markers for detecting the severity of atopic dermatitis, 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 markers of the present invention may be genes or their expression products shown in Table 1 or Table 2 below, or combinations thereof. In one embodiment, the markers of the present invention are nucleic acid markers such as the DNA of the genes or RNA that is their transcription product. In another embodiment, the markers of the present invention are protein markers that are translation products of the genes. Preferably, the markers of the present invention are nucleic acid markers, more preferably RNA markers.

[0021] [Table 1]

[0022] [Table 2]

[0023] The genes listed 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 they themselves or expression products derived therefrom function as markers for detecting the severity of atopic dermatitis. Here, "substantially identical sequences" refers to sequences that share 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 following conditions: expectation value = 10; gaps allowed; filtering = ON; match score = 1; mismatch score = -3.

[0024] As will be described 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 patients with mild to moderate atopic dermatitis compared to healthy subjects and in patients with severe atopic dermatitis compared to patients with mild to moderate atopic dermatitis, and these genes have a higher accuracy in determining severity than CCL17 (Gene ID: 6361), a gene encoding the TARC protein that is 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 is increased in mild to moderate patients compared to healthy subjects and in severe patients compared to mild to moderate patients, and these genes had better accuracy in assessing 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 individuals and those with mild to moderate atopic dermatitis, and between those with mild to moderate atopic dermatitis and those 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 genes or their expression products in a subject to detect the severity of atopic dermatitis in the subject comprises 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 a subject of the present invention is not particularly limited in terms of gender, age, race, etc., and may range 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.

[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 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 and hair, preferably skin or skin surface lipids (SSL), more preferably 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. 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 the present invention, unless otherwise specified, "skin" is a general term for the area of ​​the body surface including the epidermis, dermis, hair follicles, and tissues such as sweat glands, sebaceous glands, and other glands.

[0029] Any means used to recover or remove SSL from the skin can be used to collect SSL from the skin of a subject. Preferably, SSL absorbent materials, SSL adhesive materials, or devices for scraping SSL from the skin, as described below, can be used. The SSL absorbent materials or SSL adhesive materials are not particularly limited as long as they have affinity for SSL, and examples include polypropylene and pulp. More detailed examples of procedures for collecting SSL from the skin include methods of absorbing SSL into sheet-like materials such as oil blotting paper or oil blotting film, methods of adhering SSL to glass plates, tape, etc., and methods of scraping SSL with a spatula or scraper. Examples of methods include scraping off and recovering the SSL using a razor or the like. To improve SSL adsorption, SSL absorbent materials pre-soaked with a highly lipophilic solvent may be used. On the other hand, 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. The skin 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 facial skin, are preferred. Furthermore, the skin site from which SSL is collected may be either a rash site with atopic dermatitis or a non-rash site without atopic dermatitis. Preferably, the rash site or a non-rash site near the rash site is preferred. Here, "near the rash site" refers to an area within 10 cm adjacent to the rash site.

[0030] The RNA-containing SSL collected from a subject may be stored for a certain period of time. To minimize degradation of the RNA contained therein, the collected SSL is preferably stored 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°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 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.

[0031] 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.

[0032] In a preferred embodiment of the method of the present invention, SSL is used as the biological sample. In this 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 ammonium 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 a reverse transcription reagent kit can be used. Preferably, a highly accurate and efficient reverse transcriptase or a reverse transcription reagent kit is used, such as M-MLV Reverse Transcriptase and its variants, or a commercially available reverse transcriptase or reverse transcription reagent kit, for example, the PrimeScript (registered trademark) Reverse Transcriptase series (Takara Bio Inc.), Super rScript (registered trademark) Reverse Transcriptase series (Thermo Scientific), SuperScript (registered trademark) III Reverse Transcriptase, SuperScript ( A kit such as VILO cDNA Synthesis kit (registered trademark) (both manufactured by Thermo Scientific) is 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 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.

[0035] 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 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 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.

[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 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.

[0037] The purified PCR reaction product may be subjected to further processing 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 removal of the PCR primer sequence and adapter ligation, and the resulting reaction product may be prepared in an appropriate buffer solution. The resulting cDNA can be amplified as needed to prepare a library for quantitative analysis. These operations can be performed using, for example, the 5×VILO RT Reaction Mix included in the SuperScript® VILO cDNA Synthesis kit (Life Technologies Japan, Inc.), the 5×Ion AmpliSeq HiFi Mix included in the Ion AmpliSeq Transcriptome Human Gene Expression Kit (Life Technologies Japan, Inc.), and the 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.

[0038] 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.

[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 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.

[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, 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).

[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 probes or primers used in the above measurement, i.e., primers for specifically recognizing and amplifying the target gene of the present invention or nucleic acid derived therefrom, or probes for specifically detecting said RNA or nucleic acid derived therefrom, fall under this category. It can be designed based on the base sequence constituting the target gene. Here, "specifically recognize" means that the detected substance or product can be determined to be the target gene of the present invention or a nucleic acid derived therefrom, for example, in Northern blotting, so that substantially only the target gene of the present invention or a nucleic acid derived therefrom can be detected, or, for example, in RT-PCR, so that 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 base sequence has an 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 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 synthetic or natural. Furthermore, the probe used for hybridization is usually labeled.

[0043] 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), one-hybrid method (PNAS 100, 12271-12276 (2003)), or two-hybrid method (Biol. Reprod. 58, 302-311 (1998)) can be used. It can be used and can be selected appropriately 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, 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 labeled with a radioisotope, fluorescent substance, enzyme, or the like is used as the secondary antibody to label the primary antibody, and the signal derived from this label is measured using a radiation measuring instrument, 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)).

[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 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 mild to moderate patients, and in mild to moderate patients when detecting severe patients. The expression levels in healthy individuals, mild to moderate patients, and severe 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, 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] Furthermore, 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 can be determined appropriately based on statistical values ​​such as the average value and standard deviation of the expression levels obtained in advance as reference data of the expression levels of the target gene or its expression product in healthy subjects, mildly affected patients, moderately affected patients, and severely affected patients.

[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. In an 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 on the ROC curve, the threshold value at the point (Youden index) where "true positive rate (sensitivity)" - "false positive rate (1 - specificity)" is maximized can be used as the cutoff value (reference value).

[0047] For example, for the genes shown in Table 1, a cutoff value (reference value) for distinguishing between two groups of different severity is determined in advance, and if the expression level of the 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, and if the expression level is equal to or lower than the reference value, the group is determined to be more severe.

[0048] Furthermore, for example, cutoff values ​​(reference values) for distinguishing between two groups of different severity are 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 equal to or lower 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 higher than 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 distinguish 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 mean values ​​(AUC3) of the areas under the curve (AUC1 and AUC2) of the ROC curves for distinguishing between each of the two groups were all higher than the AUC3 obtained when the CCL17 gene encoding the TARC protein, which is known as a severity marker, was used, demonstrating high accuracy of discrimination. As mentioned 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 maximized with respect to the "true positive rate (sensitivity)" and "false positive rate (1 - specificity)" shown on the ROC curve. Examples of reference values ​​for distinguishing between two groups, healthy subjects and those with mild to moderate atopic dermatitis, and between those with mild to moderate atopic dermatitis and those with severe atopic dermatitis, using 16 genes are shown in Table 3, but the reference values ​​are not limited to these and can be determined as appropriate.

[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 predetermined 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 associated with the severity of atopic dermatitis or the score value associated with the severity of atopic dermatitis) and the expression level of the target gene or its expression product. For example, a population can be divided into multiple groups with different severity levels based on the severity of atopic dermatitis, and a reference value for determining whether or not a subject belongs to each group can be determined based on statistical values ​​(e.g., average values) of the expression level of the target gene or its expression product in each group. When multiple markers are used as the target gene or its expression product, it is preferable to determine a reference value for each marker. The population may be a group of patients with atopic dermatitis, a group combining healthy subjects and patients with atopic dermatitis, or a group of patients with atopic dermatitis of a particular severity. Furthermore, groups may be created by age, generation, gender, or race depending on 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 methods for setting the reference values ​​and classifying the severity based on the reference values ​​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 indicates a decrease in the severity of the subject's atopic dermatitis, while a decrease in its expression level over time indicates 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 previous measurement is used as the reference value. If the expression level of the negative marker measured in a subject is higher than the reference value, the severity of the subject's atopic dermatitis is detected as decreased, while if the expression level of the negative marker measured in a 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 using conventional methods during the previous measurement. In this case, if the expression level of the negative marker measured in a 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 previous measurement.

[0055] When a positive marker is used, an increase in its expression level over time indicates an increase in the severity of the subject's atopic dermatitis, while a decrease in its expression level over time indicates 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 previous measurement is used as the reference value. If the expression level of the positive marker measured in a subject is higher than the reference value, the severity of the subject's atopic dermatitis is detected as increased, while if the expression level of the positive marker measured in a subject is lower than the reference value, the severity of the subject's atopic dermatitis is detected as decreased. If necessary, the severity of the subject's atopic dermatitis may be determined using conventional methods during the previous measurement. In this case, if the expression level of the positive marker measured in a subject is higher or lower than the reference value, the severity of the subject's atopic dermatitis can be detected as higher or lower than the severity of the atopic dermatitis in the previous measurement.

[0056] In one embodiment of the method of the present invention, if the expression level of a target gene or its expression product derived from a subject is preferably 91% or less, more preferably 83% or less, and even more preferably 77% or less of the reference value, the expression level of the target gene or its expression product can be determined to be lower than the reference value. If the expression level of a target gene or its expression product derived from a subject is preferably 110% or more, more preferably 120% or more, and even more preferably 130% or more of the reference value, the expression level of the target gene or its expression product can be determined to be higher than the reference value. Alternatively, the difference between the expression level of a target gene or its expression product derived from a subject and the reference value can be determined, for example, by whether the two are statistically significantly different. When multiple markers are used as target genes or their expression products, the severity of atopic dermatitis can be detected by comparing the expression levels of each target gene or its expression product with the reference value and determining whether the expression levels of a certain percentage of the target genes or their expression products, for example, 50% or more, preferably 70% or more, more preferably 90% or more, and even preferably 100%, of the target genes or their expression products, differ from the reference 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 teacher samples of 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-to-moderate patients, moderate patients, severe patients) and healthy individuals, the expression levels of at least one or more target genes or their expression products obtained from each individual in the teacher samples as explanatory variables, and the severity of atopic dermatitis (e.g., mild, mild-to-moderate, moderate, severe) or no symptoms (healthy) for each individual as the objective variable. The expression levels of the target genes or their expression products in a 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, dimension reduction can be performed using principal component analysis (PCA), and the main components can be used as explanatory variables.

[0058] 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 RBF kernel support vector machines (SVM rbf), neural networks, generalized linear models, regularized linear discriminant analysis, and regularized logistic regression. 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.

[0059] 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).

[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, 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 a group of nine genes consisting of C1QB, DIAPH1, LCE1A, CTSV, CTSZ, KRT72, SPINK5, KRT23, and KLK5, preferably C1QB. <1> or <2> The method described below. <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 six genes consisting of LCE1A, CTSV, KRT72, SPINK5, KRT23, and KLK5, and the lower the expression level of the gene or its expression product, the more likely the subject has atopic dermatitis. Detects high severity, <1> or <2> How to do it. <5> A reference value for distinguishing between two groups with different severity levels is determined in advance, and if the expression level of the gene or its expression product of an atopic dermatitis patient is equal to or higher than the reference value, the patient is classified as a group with less severity, and if the expression level is equal to or lower than the reference value, the patient is classified as a group with more severity. <4> How to do it. <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. <1> or <2> How to do it. <7> A reference value for distinguishing between two groups with different severity levels is determined in advance, and if the expression level of the gene or its expression product in an atopic dermatitis patient is below the reference value, the patient is classified as a group with less severity, and if the expression level is above the reference value, the patient is classified as a group with more severity. <6> How to do it. <8> The severity is one or more selected from no symptoms (healthy), mild to moderate, and severe. <1> ~ <7> Either way. <9> The two groups with different severity are either healthy and mild to moderate atopic dermatitis, or mild to moderate atopic dermatitis and severe atopic dermatitis. <5> or <7> How to do it. <10> The measured values ​​of the expression level of the target gene or its expression product in the subject are substituted into a discriminant constructed using measured values ​​of the expression level of the target gene or its expression product in patients with atopic dermatitis of different severity and the expression level of the target gene or its expression product in healthy individuals as teacher samples, thereby detecting the severity of atopic dermatitis in the subject. <1> or <2> How to do it. <11> The severity is one or more selected from mild, mild-moderate, moderate, and severe. <10> How to do it. <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 do it. <13> The algorithm used for machine learning is selected from random forest, support vector machine with linear kernel (SVM linear), support vector machine with rbf kernel (SVM rbf), neural network, generalized linear model, regularized linear discriminant analysis, and regularized logistic regression. <12> How to do it. <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 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 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 selected from nine genes consisting of C1QB, DIAPH1, LCE1A, CTSV, CTSZ, KRT72, SPINK5, KRT23, and KLK5. At least one gene selected from the group of genes or its expression product, preferably C1QB or its expression product; <18> Use of. <20> The gene or its expression product is mRNA derived from lipids on the skin surface 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. [Example]

[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 in patients with mild to moderate atopic dermatitis Fourteen healthy subjects (ages 25-57, male) and 29 adults with atopic dermatitis (AD) (ages 23-56, male) were enrolled. The atopic dermatitis patients had been diagnosed by a dermatologist with mild to moderate atopic dermatitis. The head and neck EASI score (Hanifin et al., Exp Dermatol. 10, 2001) was used for diagnosis, and the patients' head and neck EASI scores ranged from 0.1 to 1.8 (mean 0.50). Sebum was collected from each subject's entire face using a single piece of oil-blotting film (polypropylene, 5.0 cm x 8.0 cm, 3M). The oil-blotting film was then transferred to a vial and stored at -80°C for approximately one month before use in RNA extraction.

[0062] 2) Diagnosis and SSL collection in patients with severe atopic dermatitis Thirty healthy subjects (ages 24-59, male) and 30 adults with atopic dermatitis (AD) (ages 21-59, male) were enrolled. The atopic dermatitis patients had been diagnosed with severe atopic dermatitis by a dermatologist. The head and neck EASI score (Hanifin et al., Exp Dermatol. 10, 2001) was used for diagnosis, and the patients' head and neck EASI scores ranged from 2.4 to 6.6 (mean, 3.0). Sebum was collected from each subject's entire face using a single piece of oil-blotting film (polypropylene, 5.0 cm x 8.0 cm, 3M). The oil-blotting film was then transferred to a vial and stored at -80°C for approximately one month before use in RNA extraction.

[0063] 3) RNA preparation and sequencing The oil-blotting film used to collect sebum in steps 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 with an RNA extraction spin column according to the attached protocol. The extracted RNA was reverse-transcribed at 42°C for 90 minutes using a 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 resulting cDNA by multiplex PCR. Multiplex PCR was performed using an 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 then transferred to Ion 540 The resulting reads were loaded onto a chip and sequenced using the Ion S5 / XL system (Life Technologies Japan). Each read sequence was then matched to the hg19 AmpliSeq Transcriptome, a reference sequence for the human genome. The gene from which each read sequence originated was determined by gene mapping using tome ERCC v1.

[0064] 4) Data analysis: Gene expression changes according to severity of head and neck EASI score i) Usage Data The SSL-derived RNA expression data (read count values) from a total of 44 healthy individuals, 29 mild-to-moderate AD patients, and 30 severe AD patients measured in 3) above were corrected using a method called DESeq2. However, only 7,177 genes for which non-missing expression data was obtained in 90% or more of the total sample subjects were used in the following analysis. Count values ​​corrected using the DESeq2 method (normalized count values) were used for the analysis.

[0065] ii) RNA expression analysis 1 Based on the normalized counts of SSL-derived RNAs measured in i) above, we identified RNAs (differentially expressed genes) with a p-value corrected by likelihood ratio (FDR) of less than 0.1 in mild to moderate AD compared to healthy controls. As a result, 1,424 RNAs were found to be downregulated in mild to moderate AD, and 1,966 RNAs were found to be upregulated in mild to moderate AD.

[0066] iii) RNA expression analysis 2 Based on the normalized counts of SSL-derived RNAs in mild to moderate AD and severe AD measured in i) above, we identified RNAs (differentially expressed genes) whose p-values ​​(FDR) corrected by likelihood ratio test were less than 0.1 in severe AD compared to mild to moderate AD. As a result, 1,436 RNAs were decreased (DOWN) in severe AD, and 1,417 RNAs were increased (UP) in severe AD.

[0067] iv) Identification of genes whose expression gradually decreases with the severity of the disease Genes corresponding to both the 1,424 genes whose expression was reduced in mild to moderate AD compared to healthy individuals, as identified in ii) above, and the 1,436 genes whose expression was reduced in severe AD compared to mild to moderate AD, as identified in iii), were extracted, and 74 genes were found to be relevant. These 74 genes are genes whose expression may be gradually reduced according to the severity of AD, that is, genes whose expression level can be used to detect the severity of atopic dermatitis. Among these genes, the related Reactome Pathways and GO terms were identified. Based on information on the genes that make up the epidermis as well as genes reported in literature that would be known to those skilled in the art, 13 genes were extracted as genes related to epidermal function.Then, from these 13 genes as candidates, genes with higher discrimination accuracy were selected in the evaluation in the following 5).

[0068] v) Identification of genes whose expression increases gradually with the severity of the disease When 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, 34 genes were found to be included. These 34 genes are genes whose expression may increase gradually with the severity of AD, that is, genes whose expression level can be used to detect the severity of atopic dermatitis. Among these genes, the related Reactome Pathways and GO terms were identified. Based on information on the genes that make up the disease, as well as genes reported in literature known to those skilled in the art, nine genes related to immune function were extracted. Among these was CCL17, a gene that encodes 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 discriminatory accuracy in the evaluation in the following step 5).

[0069] 5) Selection of more accurate markers for determining severity i) Distinguishing between healthy individuals and mild to moderate AD The accuracy of discriminating between healthy and mild-moderate AD was evaluated using ROC curves for the 22 genes extracted in iv) and v) of 4) above. ROC curves are used to discriminate between negative and positive samples using a continuous value as an index. The vertical axis plots the probability of a positive result in the positive group (sensitivity or true positive rate), while the horizontal axis plots the probability of a negative result in the negative group (specificity) minus 1 (false positive rate). The larger the area under the curve (AUC) of the ROC curve, the higher the accuracy of discriminating between the two groups. For the evaluation of the accuracy of discriminating between healthy and mild-moderate AD using the 13 genes in iv) above, mild-moderate AD was designated the negative group, and healthy individuals were designated the positive group. For the evaluation of the accuracy of discriminating between healthy and mild-moderate AD using the 9 genes in v) above, healthy individuals were designated the negative group, and mild-moderate AD was designated 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 was evaluated using ROC curves for the 22 genes extracted in 4) iv) and v) above. In evaluating the accuracy of discriminating between mild and moderate AD using the 13 genes in iv) above, severe AD was classified as the negative group, and mild and moderate AD was classified as the positive group. In evaluating the accuracy of discriminating between mild and moderate AD using the 9 genes in v) above, mild and moderate AD was classified as the negative group, and severe AD was classified as the positive group. The accuracy rate (accuracy rate 2) of the Youden index, the AUC value (AUC2) at that time, and the cutoff value (reference value 2) were calculated for each of the 22 genes.

[0071] iii) Selection of genes with higher accuracy suitable for distinguishing between healthy individuals, mild to moderate AD, and severe AD The average AUC3 (AUC1 and AUC2) of the 22 genes extracted in 4) above was used to evaluate whether they could accurately distinguish between healthy controls and mild-moderate AD, and between mild-moderate AD and severe AD (Table 3). The AUC3 (AUC3 = 0.693) of CCL17, a gene encoding the TARC protein known to reflect the severity of AD, was used as a comparison, and genes with a higher AUC3 were extracted. As a result, 11 genes were extracted from the 13 genes extracted in 4) iv) above: LCE1A, CTSV, KRT72, SPINK5, KRT23, KLK5, LCE1B, SPRR1A, PKP1, KRT74, and KRT25. Furthermore, five genes were extracted from the nine genes extracted in 4) v) above: C1QB, DIAPH1, CTSZ, APRT, and S100B. The AUC1, accuracy rate 1, reference value 1, AUC2, accuracy rate 2, reference value 2, and AUC3 of the 16 extracted genes and the comparison target CCL17 are shown in Table 3. In the table, "decrease" in expression changes indicates that expression decreases with worsening severity, and "increase" indicates that expression increases with worsening severity. From the above results, the AUC3 of the 16 genes consisting of C1QB, DIAPH1, LCE1A, CTSV, CTSZ, KRT72, SPINK5, KRT23, KLK5, LCE1B, SPRR1A, PKP1, KRT74, APRT, KRT25, and S100B is higher than that of CCL17, and therefore these genes are considered to be capable of distinguishing between healthy individuals and mild to moderate AD, and between mild to moderate AD and severe AD with high accuracy.

[0072] [Table 3]

Claims

[Claim 1] A method for measuring the expression level of gene mRNA in a subject, comprising a step of measuring the expression level of SPINK5 mRNA in lipids on the skin surface collected from the entire face of the subject in order to detect the severity of atopic dermatitis in the subject, the method comprising: determining in advance a reference value 1 for distinguishing between a healthy group and a mild-to-moderate group, and a reference value 2 for distinguishing between the mild-to-moderate group and a severe group; and indicating that if the expression level of SPINK5 mRNA in an atopic dermatitis patient is equal to or greater than reference value 1, the patient is healthy; if the expression level is less than reference value 1 and equal to or greater than reference value 2, the patient is mild to moderate; and if the expression level is less than reference value 2, the patient is severe.

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