Method for detecting severity of atopic dermatitis

Abstract

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

Description

Technical Field

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

Background Art

[0002] Atopic dermatitis (hereinafter also referred to as "AD") is an eczematous skin disease that mainly develops in individuals with an atopic predisposition. Typical symptoms of atopic dermatitis include chronic and recurrent itching, rashes, erythema, etc. that occur bilaterally, as well as parakeratosis, reduced barrier function, dry skin, etc. Many cases of atopic dermatitis develop in infants and tend to improve with growth, but in recent years, adult-type and refractory atopic dermatitis have also been increasing.

[0003] The presence or absence 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, as a method for objectively determining the severity of atopic dermatitis, a test method using the blood TARC (thymus and activation-regulated chemokine) value or SCCA2 (Squamous cell carcinoma antigen 2) value as an index has been used. These are used as indices that sensitively reflect the disease state of atopic dermatitis, and are used not only to evaluate the presence or absence and severity of atopic dermatitis, but also for patient education and determination of treatment policies (see Non-Patent Documents 1, 2, and 3). However, the test method using the blood TARC value or SCCA2 value as an index requires blood sampling, so it is an invasive method. Furthermore, there are problems such as the need for time until the results are obtained.

[0004] Meanwhile, technologies are being 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 advantages such as the availability of comprehensive analysis methods, the ability to obtain abundant information in a single analysis, and the ease of functionally linking the analysis results based on numerous research reports on single nucleotide polymorphisms and RNA function. Nucleic acids derived from living organisms can be extracted from bodily fluids such as blood, secretions, and tissues, but recently it has been reported that RNA contained in skin surface lipids (SSL) can be used as a 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 Public Gazette No. 2018 / 008319 [Patent Document 2] Japanese Patent Publication No. 2020-74769 [Non-patent literature]

[0006] [Non-Patent Document 1] Sugawara et al., Allergy (2002) 57:180-181: [Non-Patent Document 2] Ohta et al., Ann Clin Biochem.(2012) 49:277-84 [Non-Patent Document 3] Kato et al., Japanese Journal of Dermatology (2018) 128: 2431-2502 [Overview of the Initiative] [Problems that the invention aims to solve]

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

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

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

[0010] According to the present invention, it is possible to detect the severity of atopic dermatitis early, accurately, and objectively in a simple and non-invasive manner. [Modes for carrying out the invention]

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

[0012] In this invention, "gene" means a double-stranded DNA including human genomic DNA, as well as single-stranded DNA (positive strand) including cDNA, single-stranded DNA (complementary strand) having a sequence complementary to the positive strand, and fragments thereof, in which the sequence information of the bases constituting the DNA contains some kind of biological information. Furthermore, the term "gene" in question encompasses not only genes represented by a specific base sequence, but also nucleic acids encoding their homologs (i.e., orthologs), variants such as genetic polymorphisms, and derivatives. The gene names disclosed herein follow the Official Symbols listed on the NCBI ([www.ncbi.nlm.nih.gov / ]).

[0013] In this invention, the term "expression product" of a gene is a concept that encompasses both the transcript and the translation product of a gene. A "transcript" is RNA produced by transcription from a gene (DNA), and a "translation product" is a protein encoded by a gene that is synthesized through translation based on RNA.

[0014] In this invention, "atopic dermatitis" refers to a disease characterized by itchy eczema that repeatedly worsens and remits, and most patients with this disease are considered to have an atopic predisposition. Atopic predispositions include i) a family history or past medical history (one or more of the following diseases: bronchial asthma, allergic rhinitis / conjunctivitis, atopic dermatitis), or ii) a predisposition to easily produce IgE antibodies.

[0015] In this invention, "severity of atopic dermatitis" refers to the degree of progression of atopic dermatitis, and is classified according to the condition of the skin lesions, for example, asymptomatic, mild, moderate, and severe. The characteristics of the rash include erythema, edema / inflammation / papules (solid, serous), exudate / crust, scratch marks, lichenification, dryness, itching, prurigo nodules, scales (branny, leaf-shaped, membranous, etc.), blisters, pustules, erosions, ulcers, etc. As for the severity classification, 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.>). In addition to these, there are the atopic dermatitis severity classification by the Atopic Dermatitis Severity Classification Study Committee of the Japanese Dermatological Association (Journal of the Japanese Dermatological Association <2001; 111: 2023-2033, Journal of the Japanese Dermatological Association, 1998; 108: 1491-1496.>), Severity Scoring of Atopic Dermatitis (「SCORAD」<Dermatology, 1993; 186: 23-31.>, etc.) that are known.

[0016] For example, EASI uses the head and neck, trunk, upper limbs, and lower limbs as evaluation sites, and is calculated based on the scores for four symptoms of erythema, edema / inflammation / papules, scratch marks, and lichenification at each evaluation site, and the percentage (%) of the area of the above four symptoms in the entire evaluation site, and is a value from 0 to 72. Also, in IGA, the physician or researcher who is the evaluator comprehensively evaluates the state of the rash for the whole body or specific evaluation sites, and gives a five-level evaluation of "no symptoms", "mild", "moderate", "severe", and "very severe". In some cases, it may be a six-level evaluation including "most severe". The IGA score can be converted into an ordinal scale such as "no symptoms = 0", "mild = 1", "moderate = 2", "severe = 3", "very severe = 4", and "most severe = 5" and used.

[0017] In the detection of the severity of atopic dermatitis of the present invention, it is possible to detect "no symptoms", "mild", "moderate", and "severe", but it is preferable to detect "no symptoms", "mild", and "moderate". In the present invention, "mild" refers to a condition mainly characterized by dryness and mild erythema, scale layer, etc., and corresponds to an EASI score greater than 0 and less than 6, or an IGA score of 2. "Moderate" refers to a condition mainly characterized by moderate erythema, scale layer, a small number of papules, scratch marks, etc., and corresponds to an EASI score of 6 or more and less than 23, or an IGA score of 3. "Severe" refers to a condition mainly characterized by erythema accompanied by severe swelling / edema / infiltration or lichenification, multiple papules, severe scaling, adhesion of crusts, small blisters, erosions, a large number of scratch marks, prurigo nodules, etc., and corresponds to an EASI score of 23 or more and 72 or less, or an IGA score of 4. "No symptoms" means remission or almost remission, with no symptoms and at the same level as a healthy person. In addition, in the evaluation of "no symptoms", "mild", "moderate" and "severe" using the EASI score and IGA score, the score range serving as the criterion for each evaluation is not limited to the upper limit and can be determined as appropriate.

[0018] In the present invention, "detection" of the severity of atopic dermatitis means clarifying the severity of atopic dermatitis, and can also be paraphrased by terms such as examination, measurement, determination or evaluation support. In the present invention, the terms "detection", "examination", "measurement", "determination" or "evaluation" do not include the diagnosis of the severity of atopic dermatitis by a doctor.

[0019] As shown in the examples described below, for 14 healthy subjects and 29 atopic dermatitis patients diagnosed as mild (score: greater than 0 and less than 6) and 11 patients judged as moderate (score: 6 or more and less than 23) according to the severity of EASI, a total of 29 patients, RNA expression analysis was performed according to the following procedures 1) to 2), and the five genes of CAPN1, GRN, NCOR2, PLP2, and PPP1R9B increased in a severity-dependent manner. 1) Obtain data (read count values) of the expression levels of RNA extracted from the SSL of 14 healthy subjects and 29 atopic dermatitis patients (adults). 2) The read count values ​​are converted to RPM values ​​corrected for differences in the total number of reads between samples. The RPM+1 value, obtained by adding an integer 1 to this value, is then converted to a base-2 logarithm (Log2(RPM+1) value). Based on this value, Tukey tests are performed between the three groups (healthy individuals, mildly ill patients, and moderately ill patients), and genes for which the p-value is less than 0.1 are selected in all cases: healthy individuals vs. mildly ill patients, mildly ill patients vs. moderately ill patients, and healthy individuals vs. moderately ill patients. Here, the "p-value" in statistical testing indicates the probability that a statistic more extreme than the statistic actually calculated from the data under the null hypothesis will be observed. Therefore, the smaller the "p-value," the more significant the difference between the comparison groups is considered to be.

[0020] Furthermore, RNA expression analysis was performed on 14 healthy individuals and 29 atopic dermatitis patients (17 diagnosed as mild and 12 as moderate based on a comprehensive evaluation of 0 (no symptoms), 1 (mild), 2 (mild), 3 (moderate), and 4 (severe) facial IGA) using the same procedure as above (1) to (2). The results showed that LOC146880, PPP1R12C, and SYVN1 increased in a severity-dependent manner, while SLC31A1 decreased in a severity-dependent manner.

[0021] Therefore, genes selected from the group of nine genes consisting of CAPN1, NCOR2, PPP1R9B, PLP2, GRN, PPP1R12C, LOC146880, SLC31A1, and SYVN1, or their expression products, can serve as markers for detecting the severity of atopic dermatitis. Each of these nine genes can individually serve as a marker for detecting the severity of atopic dermatitis, but it is also possible to use two or more genes in combination. Preferably, to detect the severity of atopic dermatitis based on EASI, detection markers are selected from five genes out of the nine mentioned above: CAPN1, NCOR2, PPP1R9B, PLP2, and GRN. To detect the severity of atopic dermatitis based on IGA, detection markers are selected from four genes out of the nine mentioned above: PPP1R12C, LOC146880, SLC31A1, and SYVN1. Furthermore, these nine genes are novel markers for determining the severity of atopic dermatitis, as their relationship to the severity of atopic dermatitis has not been reported to date.

[0022] Furthermore, the genes that can serve as markers for detecting the severity of atopic dermatitis (hereinafter also referred to as "target genes") include genes that have a base sequence substantially identical to the base sequence of the DNA constituting the gene, insofar as they can serve as biomarkers for detecting the severity of atopic dermatitis. Here, substantially identical base sequences mean, for example, that when searching using the homology calculation algorithm NCBI BLAST with the conditions expected value = 10; gap allowed; filtering = ON; match score = 1; mismatch score = -3, the base sequence of the gene has 90% or more identity with the base sequence of the DNA constituting the gene, preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more.

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

[0024] In the method for detecting the severity of atopic dermatitis according to the present invention, the gender, age, and race of the subjects are not particularly limited and may include infants to the elderly. Preferably, the subjects are people who need or wish to have the severity of atopic dermatitis detected. For example, the subjects are people who have atopic dermatitis, people suspected of having atopic dermatitis, or people who have a genetic predisposition to atopic dermatitis.

[0025] The biological samples used in the present invention may be any tissue or biomaterial in which the expression of the gene of the present invention changes according to the severity of atopic dermatitis. Specifically, examples include organs, skin, blood, urine, saliva, sweat, stratum corneum, surface lipids (SSL), tissue exudates and other bodily fluids, serum and plasma prepared from blood, and others such as feces and hair. Preferably, the samples are skin, stratum corneum, or surface lipids (SSL), and more preferably, surface lipids (SSL). The site of skin from which SSL is collected is not particularly limited and may be any part of the body such as the head, face, neck, trunk, hands and feet. Areas with high sebum secretion, such as the skin of the head or face, are preferred, and facial skin is more preferred. Furthermore, the site of skin from which SSL is collected may be either a lesion area where atopic dermatitis has developed or an unaffected area, but preferably, the lesion area or an unaffected area near the lesion area is preferred. Here, "near the lesion area" refers to an area within 10 cm adjacent to the lesion area.

[0026] Here, "surface lipids (SSL)" refers to the lipid-soluble fraction present on the surface of the skin, and is sometimes called sebum. Generally, SSL mainly consists of secretions from exocrine glands such as sebaceous glands in the skin, and exists 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 above). Furthermore, in this specification, "skin" is a general term for the region including the stratum corneum, epidermis, dermis, hair follicles, and tissues such as sweat glands, sebaceous glands, and other glands, unless otherwise specified.

[0027] Any means used for the collection or removal of SSL from the skin can be employed to collect SSL from the subject's skin. Preferably, an SSL absorbent material, an SSL adhesive material, or an instrument for scraping off SSL from the skin, as described later, can be used. The SSL absorbent material or SSL adhesive material is not particularly limited as long as it is a material that has an 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 onto a sheet material such as oil-blotting paper or oil-blotting film, methods of adhering SSL to a glass plate or tape, and methods of scraping off and collecting SSL with a spatula, scraper, etc. To improve the adsorption of SSL, an SSL absorbent material containing a highly lipid-soluble solvent beforehand may be used. On the other hand, since the adsorption of SSL is inhibited if the SSL absorbent material contains a highly water-soluble solvent or water, it is preferable that the content of highly water-soluble solvents or water is low. It is preferable to use the SSL absorbent material in a dry state.

[0028] RNA-containing SSLs collected from subjects may be stored for a certain period of time. To minimize the degradation of the contained RNA, it is preferable to store the collected SSLs under low-temperature conditions as quickly as possible after collection. The storage temperature conditions for the RNA-containing SSLs in this invention may be 0°C or lower, preferably -20±20°C to -80±20°C, more preferably -20±10°C to -80±10°C, even more preferably -20±20°C to -40±20°C, even more preferably -20±10°C to -40±10°C, even more preferably -20±10°C, and even more preferably -20±5°C. The storage period for the RNA-containing SSLs under these 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, and even more preferably 3 months or less, for example, 3 days to 3 months.

[0029] In the present invention, the objects to be measured for the expression level of a target gene or its expression product include cDNA artificially synthesized from RNA, the DNA encoding that RNA, the protein encoded by that RNA, molecules that interact with that protein, molecules that interact with that RNA, or molecules that interact with that DNA. Here, molecules that interact with RNA, DNA, or proteins include DNA, RNA, proteins, polysaccharides, oligosaccharides, monosaccharides, lipids, fatty acids, and their phosphorylated, alkylated, and glycosidic compounds, as well as complexes of any of the above. Furthermore, the expression level comprehensively refers to the amount of expression or activity of the gene or expression product in question.

[0030] In the method of the present invention, in a preferred embodiment, 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 amplified product is measured. For RNA extraction from SSL, methods commonly used for RNA extraction or purification from biological samples can be employed, such as the phenol / chloroform method, the AGPC (acid guanidinium thiocyanate-phenol-chloroform extraction) method, or methods using columns such as TRIzol®, RNeasy®, or QIAzol®, or methods using special silica-coated magnetic particles, methods using Solid Phase Reversible Immobilization magnetic particles, or extraction using commercially available RNA extraction reagents such as ISOGEN.

[0031] For the reverse transcription, primers targeting a specific RNA to be analyzed may be used, but for more comprehensive nucleic acid preservation and analysis, random primers are preferable. A general reverse transcriptase or reverse transcription reagent kit can be used for the reverse transcription. Preferably, a reverse transcriptase or reverse transcription reagent kit with high accuracy and efficiency is used, such as M-MLV Reverse Transcriptase and its variants, or commercially available reverse transcriptase or reverse transcription reagent kits, such as the PrimeScript® Reverse Transcriptase series (Takara Bio Inc.) and the SuperScript® Reverse Transcriptase series (Thermo Scientific Inc.). SuperScript® III Reverse Transcriptase and SuperScript® VILO cDNA Synthesis kit (both from Thermo Scientific Inc.) are preferably used. In the reverse transcription extension reaction, it is preferable to adjust the temperature to preferably 42°C ± 1°C, more preferably 42°C ± 0.5°C, and even more preferably 42°C ± 0.25°C, while adjusting the reaction time to preferably 60 minutes or more, more preferably 80 to 120 minutes.

[0032] Methods for measuring expression levels can be selected from nucleic acid amplification methods such as PCR, real-time RT-PCR, multiplex PCR, SmartAmp, and LAMP, which use DNA that hybridizes to RNA, cDNA, or DNA as primers; hybridization methods (DNA chips, DNA microarrays, dot blot hybridization, slot blot hybridization, Northern blot hybridization, etc.) which use nucleic acids that hybridize to these as probes; methods for determining the base sequence (sequencing); or methods combining these.

[0033] In PCR, a primer pair targeting a specific DNA to be analyzed may be used to amplify only that specific DNA, or multiple primer pairs may be used to amplify multiple specific DNAs simultaneously. Preferably, the PCR is multiplex PCR. Multiplex PCR is a method of simultaneously amplifying multiple gene regions by using multiple primer pairs simultaneously in the PCR reaction system. Multiplex PCR can be performed using commercially available kits (for example, the Ion AmpliSeqTranscriptome Human Gene Expression Kit; Life Technologies Japan Co., Ltd., etc.). The temperature for the annealing and extension reactions in the PCR cannot be generalized as it depends on the primers used, but when using the above-mentioned multiplex PCR kit, 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 reactions are preferably performed in one step. The duration of 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 is preferably 95 to 99°C for 10 to 60 seconds. Reverse transcription and PCR at the above temperatures and times can be performed using a thermal cycler commonly used for PCR.

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

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

[0036] When measuring the expression level of a target gene or nucleic acid derived therefrom using Northern blot hybridization, for example, a probe DNA is first labeled with a radioisotope, a fluorescent substance, etc. Then, the resulting labeled DNA is hybridized with RNA derived from a biological sample transferred to a nylon membrane, etc., according to a conventional method. Subsequently, the double helix formed between the labeled DNA and RNA is measured by detecting the signal originating from the label.

[0037] When measuring the expression level of a target gene or nucleic acid derived therefrom using RT-PCR, for example, cDNA is first prepared from RNA derived from a biological sample according to a conventional method, and a pair of primers (a positive strand that binds to the cDNA (- strand), and a reverse strand that binds to the + strand) prepared to amplify the target gene of the present invention are hybridized with this cDNA as a template. Then, PCR is performed according to a conventional method, and the resulting amplified double-stranded DNA is detected. For the detection of the amplified double-stranded DNA, a method can be used to detect labeled double-stranded DNA produced by performing the above PCR using primers that have been previously labeled with an RI, a fluorescent substance, etc.

[0038] When measuring the expression level of a target gene or nucleic acid derived therefrom using a DNA microarray, for example, an array in which at least one nucleic acid (cDNA or DNA) derived from the target gene of the present invention is immobilized on a support is used, labeled cDNA or cRNA prepared from mRNA is bound to the microarray, and the expression level of mRNA can be measured by detecting the label on the microarray. The nucleic acid immobilized on the array can 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 it may be a nucleic acid consisting of a partial sequence. Here, "partial sequence" refers to a nucleic acid consisting of at least 15 to 25 bases. Here, stringent conditions can typically be washing conditions of about "1×SSC, 0.1%SDS, 37°C", more stringent hybridization conditions can be about "0.5×SSC, 0.1%SDS, 42°C", and even more stringent hybridization conditions can be about "0.1×SSC, 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.

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

[0040] The probes or primers used in the above measurements, namely primers for specifically recognizing and amplifying the target gene of the present invention or nucleic acids derived therefrom, or probes for specifically detecting the RNA or nucleic acids derived therefrom, can be designed based on the base sequence constituting the target gene. Here, "specifically recognizing" means, for example, in the Northern blotting method, that substantially only the target gene of the present invention or nucleic acids derived therefrom can be detected, or for example, in the RT-PCR method, that substantially only the nucleic acids in question are amplified, so that the detected substance or product can be determined to be the gene or nucleic acids derived therefrom. Specifically, the present invention can utilize DNA consisting of the base sequence constituting the target gene, or oligonucleotides containing a certain number of nucleotides complementary to its complementary strand. Here, "complementary strand" refers to the other strand of a double-stranded DNA consisting of A:T (U in the case of RNA) and G:C base pairs. Furthermore, "complementary" is not limited to cases where the sequence is perfectly complementary in the given number of consecutive nucleotide regions, but is preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 98% or more of the base sequence identity. The base sequence identity can be determined by algorithms such as BLAST. When used as a primer, such oligonucleotides only need to be able to perform specific annealing and chain extension, and typically have a chain length of, for example, 10 bases or more, preferably 15 bases or more, more preferably 20 bases or more, and for example 100 bases or less, preferably 50 bases or less, and more preferably 35 bases or less. When used as a probe, it is sufficient that specific hybridization can be performed, and oligonucleotides that 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 are used, and for example, oligonucleotides with a chain length of 10 bases or more, preferably 15 bases or more, and for example 100 bases or less, preferably 50 bases or less, and more preferably 25 bases or less are used. Here, "oligonucleotides" can be DNA or RNA, and may be synthetic or natural. Alternatively, the probes used for hybridization are usually labeled.

[0041] Furthermore, when measuring the translation product (protein) of the target gene of the present invention, molecules that interact with said protein, molecules that interact with RNA, or molecules that interact with DNA, methods such as protein chip analysis, immunoassay (e.g., ELISA), mass spectrometry (e.g., LC-MS / MS, MALDI-TOF / MS), 1-hybrid methods (PNAS 100, 12271-12276 (2003)), and 2-hybrid methods (Biol. Reprod. 58, 302-311 (1998)) can be used and can be appropriately selected depending on the target. For example, when a protein is used as the target of measurement, the procedure is carried out by contacting a biological sample with an antibody against the expression product of the present invention, detecting the polypeptide in the sample bound to the antibody, and measuring its level. For example, using the Western blotting method, the above antibody is used as the primary antibody, and then the primary antibody is labeled with a radioisotope, a fluorescent substance, or an enzyme, and the signal derived from these labeling substances is measured using a radiation detector, fluorescence detector, etc. Furthermore, the antibodies against the above-mentioned translation products may be polyclonal or monoclonal antibodies. These antibodies can be manufactured according to known methods. Specifically, polyclonal antibodies can be obtained by using proteins expressed and purified in E. coli or other bacteria according to conventional methods, or by synthesizing partial polypeptides of such proteins according to conventional methods, immunizing non-human animals such as rabbits, and then obtaining them from the serum of the immunized animals according to conventional methods. On the other hand, monoclonal antibodies can be obtained from hybridoma cells prepared by immunizing non-human animals such as mice with proteins expressed and purified in E. coli or other bacteria according to conventional methods, or with partial polypeptides of said proteins, and then fusing the resulting spleen cells with myeloma cells. Monoclonal antibodies may also be produced using phage display (Griffiths, AD; Duncan, AR, Current Opinion in Biotechnology, Volume 9, Number 1, February 1998, pp. 102-108(7)).

[0042] Thus, the expression level of the target gene of the present invention or its expression product in a biological sample taken from a subject is measured, and the severity of atopic dermatitis is detected based on the expression level. Specifically, the detection is 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, it is preferable to use the following as indicators, as described above: the read count value, which is the expression level data; the RPM value obtained by correcting the difference in the total number of reads between samples from the read count value; the value obtained by converting the RPM value to a base-2 logarithm (Log2RPM value) or the base-2 logarithm obtained by adding an integer 1 (Log2(RPM+1) value); or the count value corrected using DESeq2 (Love MI et al. Genome Biol. 2014) (Normalized count value) or the base-2 logarithm obtained by adding an integer 1 (Log2(count+1) value). Alternatively, values ​​calculated using common RNA-seq quantitative values ​​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 be used. Furthermore, signal values ​​obtained by microarray methods and their corrected values ​​may also be used. Furthermore, when analyzing only specific target genes using methods such as RT-PCR, it is preferable to either convert the expression level of the target gene to a relative expression level based on the expression level of housekeeping genes for analysis, or to quantify the absolute copy number (absolute quantification) using a plasmid containing the region of the target gene for analysis. The copy number obtained by digital PCR may also be used. Here, "control level" refers to, for example, the expression level of the target gene or its expression product in healthy individuals when detecting mild cases, or in mild cases when detecting moderate cases. The expression levels in healthy individuals and mild cases may be statistical values ​​(e.g., mean values) of the expression levels of the gene or its expression product measured from a population of healthy individuals and mild cases. If there are multiple target genes, it is preferable to determine the reference expression level for each gene or its expression product.

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

[0044] Furthermore, by utilizing the measured expression levels of target genes or their expression products derived from atopic dermatitis patients with varying degrees of severity, and the measured expression levels of target genes or their expression products derived from healthy individuals, a discriminant formula (predictive model) can be constructed to differentiate between patient groups with varying degrees of severity (e.g., mild, moderate, and severe cases) and healthy individuals (asymptomatic). This discriminant formula can then be used to detect the severity of atopic dermatitis. Specifically, using the measured expression levels of target genes or their expression products derived from atopic dermatitis patients with varying degrees of severity, and the measured expression levels of target genes or their expression products derived from healthy individuals, as training samples, a discriminant formula (predictive model) is constructed to differentiate between patient groups with varying degrees of severity (e.g., mild, moderate, and severe cases) and healthy individuals (asymptomatic). Based on this discriminant formula, a cutoff value (reference value) is determined to differentiate between each patient group with varying degrees of severity. Note that in creating the discriminant formula, dimensionality reduction can be performed using principal component analysis (PCA), and the major components can be used as explanatory variables. Then, by similarly measuring the level of the target gene or its expression product from biological samples taken from the subject, substituting the obtained measurement values ​​into the discriminant formula, and comparing the results obtained from the discriminant formula with a reference value, the severity of atopic dermatitis in the subject can be detected.

[0045] For constructing discriminant formulas, publicly known algorithms such as those used in machine learning can be utilized. Examples of machine learning algorithms include Random Forest and Linear Kernel Support Vector Machine (SVM). Examples include linear, rbf kernel support vector machine (SVM rbf) neural networks, generalized linear models, regularized linear discriminant analysis, and regularized logistic regression. By inputting validation data into the constructed prediction model and calculating predicted values, the model that best matches the predicted values ​​to the observed values, for example, the model with the highest accuracy, can be selected as the optimal prediction model. Additionally, the recall rate, precision, and their harmonic mean (F-score) can be calculated from the predicted and observed values, and the model with the highest F-score can be selected as the optimal prediction model.

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

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

[0048] With regard to the embodiments described above, the present invention further discloses the following embodiments. <1> A method for detecting the severity of atopic dermatitis in a subject, comprising the step of measuring the expression level of at least one gene or its expression product selected from a group of nine genes consisting of CAPN1, NCOR2, PPP1R9B, PLP2, GRN, PPP1R12C, LOC146880, SLC31A1, and SYVN1, in a biological sample taken from the subject. <2> The severity is based on the EASI score or the IGA score. <1> The method. <3> The severity is based on the EASI score, and the expression level measurement is the measurement of the expression level of at least one gene or its expression product selected from a group of five genes consisting of CAPN1, NCOR2, PPP1R9B, PLP2, and GRN. <2> The method. <4> The severity is based on the IGA score, and the expression level measurement is the measurement of the expression level of at least one gene or its expression product selected from a group of four genes consisting of PPP1R12C, LOC146880, SLC31A1, and SYVN1. <2> The method. <5> The severity level is one or more, selected from asymptomatic, mild, and moderate. <1> ~ <4> One of the following methods. <6> The expression level of a gene or its expression product is measured by the amount of mRNA expression. <1> ~ <5> One of the following methods. <7> The gene or its expression product is RNA contained in the lipids on the skin surface of the subject. <1> ~ <6> One of the following methods. <8> The severity of atopic dermatitis is detected by comparing the measured expression levels with the reference values ​​of each gene or its expression product. <1> ~ <7> One of the following methods. <9> Use of at least one gene or its expression product, selected from a group of nine genes consisting of CAPN1, NCOR2, PPP1R9B, PLP2, GRN, PPP1R12C, LOC146880, SLC31A1, and SYVN1, derived from biological samples taken from subjects, as a severity marker for atopic dermatitis. <10> The use of at least one gene or its expression product selected from a group of five genes consisting of CAPN1, NCOR2, PPP1R9B, PLP2, and GRN, and the severity is based on the EASI score. <9> Use. <11> The use of at least one gene or its expression product selected from a group of four genes consisting of PPP1R12C, LOC146880, SLC31A1, and SYVN1, and the severity is based on the IGA score. <9> Use. <12> The gene or its expression product is mRNA contained in the surface lipids of the skin collected from the subject. <9> ~ <11> Use of either of the following. <13> The system contains an oligonucleotide that specifically hybridizes with the gene or nucleic acid derived therefrom, or an antibody that recognizes the expression product of the gene. <1> ~ <8> A test kit for detecting the severity of atopic dermatitis, used in one of the following methods. <14> A marker for detecting the severity of atopic dermatitis, comprising at least one gene selected from a group of nine genes consisting of CAPN1, NCOR2, PPP1R9B, PLP2, GRN, PPP1R12C, LOC146880, SLC31A1, and SYVN1, or its expression product. [Examples]

[0049] The present invention will be described in more detail below based on examples, but the present invention is not limited thereto. Example 1: Detection of atopic dermatitis severity using RNA extracted from SSL 1) Diagnosis and SSL collection of patients with atopic dermatitis The study included 14 healthy males (25-57 years old) and 29 adult men with atopic dermatitis (AD) (23-56 years old). Patients with atopic dermatitis had been diagnosed with mild or moderate atopic dermatitis by a dermatologist. The EASI score (Hanifin et al. Exp dermatol. 10, 2001) was used for diagnosis, and according to the literature, patients with a score greater than 0 but less than 6 were classified as having mild atopic dermatitis, and patients with a score of 6 or greater but less than 23 were classified as having moderate atopic dermatitis (Chopra et al. Br J Dermatol. 177, 2017). As a result, there were 18 patients with mild atopic dermatitis and 11 patients with moderate atopic dermatitis. Furthermore, the facial areas of the above-mentioned atopic dermatitis patients were comprehensively evaluated using the IGA score on a 5-point scale: 0 (no symptoms), 1 (very mild), 2 (mild), 3 (moderate), and 4 (severe). As a result, 17 patients had a mild case with a score of 2, and 12 patients had a moderate case with a score of 3. Sebum was collected from the entire face of each subject using an oil-absorbing film, and the oil-absorbing film was transferred to a vial and stored at -80°C for approximately one month until it was used for RNA extraction.

[0050] 2) RNA preparation and sequencing The oil-absorbing film described in 1) above was cut to an appropriate size, and RNA was extracted using QIAzol Lysis Reagent (Qiagen) according to the included protocol. Based on the extracted RNA, cDNA was synthesized by reverse transcription at 42°C for 90 minutes using the SuperScript VILO cDNA Synthesis kit (Life Technologies Japan Co., Ltd.). Random primers included in the kit were used as primers for the reverse transcription reaction. From the obtained cDNA, a library containing DNA derived from the 20802 gene was prepared by multiplex PCR. Multiplex PCR was performed using the Ion AmpliSeqTranscriptome Human Gene Expression Kit (Life Technologies Japan Co., Ltd.) under the conditions [99°C, 2 min → (99°C, 15 sec → 62°C, 16 min) × 20 cycles → 4°C, Hold]. The obtained PCR products were purified with Ampure XP (Beckman Coulter, Inc.), followed by buffer reconstitution, primer sequence digestion, adapter ligation and purification, and amplification to prepare the library. The prepared library was loaded onto an Ion 540 Chip and sequenced using the Ion S5 / XL system (Life Technologies Japan Co., Ltd.).

[0051] 3) Data analysis 1: Variable genes dependent on severity classification by EASI score i) Data used The RNA expression data (read count values) from subjects measured in 2) above were converted to RPM values ​​corrected for differences in the total number of reads between samples. However, only 7429 genes for which expression data without missing values ​​was obtained for more than 90% of all samples were used for the following analysis. To approximate the RPM values, which follow a negative binomial distribution, to a normal distribution, the base-2 logarithm (Log2(RPM+1) value) was used by adding the integer 1. Next, the samples were divided into three groups: healthy individuals, mildly ill patients, and moderately ill patients, according to the severity classification based on the EASI score evaluated in 1) above.

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

[0053] [Table 1-1]

[0054] [Table 1-2]

[0055] 4) Data Analysis 2: Variable genes dependent on severity classification by comprehensive facial assessment (IGA score) i) Data used The RNA expression data (read count values) from subjects measured in 2) above were converted to RPM values ​​corrected for differences in the total number of reads between samples. However, only 7429 genes for which expression data without missing values ​​was obtained for more than 90% of all samples were used for the following analysis. To approximate the RPM values, which follow a negative binomial distribution, to a normal distribution, the base-2 logarithm (Log2(RPM+1) value) was used by adding the integer 1. Next, the samples were divided into three groups: healthy individuals, mildly ill patients, and moderately ill patients, according to the severity classification based on the comprehensive facial assessment (IGA score) evaluated in 1) above.

[0056] ii) RNA expression analysis Based on the SSL-derived RNA expression levels (Log2(RPM+1) values) measured in healthy individuals, mildly ill patients, and moderately ill patients as described in i) above, Tukey assays were performed between the three groups, and genes with p-values ​​less than 0.1 were selected in all cases: healthy vs. mildly ill patients, mildly ill patients vs. moderately ill patients, and healthy vs. moderately ill patients. As a result, the expression of the three genes LOC146880, PPP1R12C, and SYVN1 increased in a severity-dependent manner, while the expression of SLC31A1 decreased in a severity-dependent manner (Table 2).

[0057] [Table 2-1]

[0058] [Table 2-2]

Claims

1. A method for detecting the severity of atopic dermatitis in a subject, comprising measuring the expression level of mRNA of at least one gene selected from a group of five genes consisting of CAPN1, NCOR2, PPP1R9B, PLP2 and GRN in surface skin lipids collected from the entire face of the subject, wherein the severity is based on the EASI (Eczema Area and Severity Index) score.

2. A method for detecting the severity of atopic dermatitis in a subject, comprising measuring the expression level of mRNA of at least one gene selected from a group of four genes consisting of PPP1R12C, LOC146880, SLC31A1 and SYVN1 in skin surface lipids collected from the entire face of the subject, wherein the severity is based on the IGA (Investigator's Global Assessment) score.

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

4. A method according to any one of claims 1 to 3, wherein the detection comprises comparing a measured value of the expression level with a reference value of the expression level of mRNA of each of the genes to detect the severity of the atopic dermatitis.