Method for detecting infantile atopic dermatitis

Abstract

To provide a method for detecting infantile atopic dermatitis using a skin surface lipid.SOLUTION: The method for detecting infantile atopic dermatitis in a subject includes a step for measuring an expression level of TARC gene or an expression product thereof in a skin surface lipid collected from the subject.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for detecting infantile atopic dermatitis using a marker for infantile atopic dermatitis derived from skin surface lipids.

Background Art

[0002] Atopic dermatitis (hereinafter also referred to as "AD") is an eczematous skin disease that mainly occurs 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. Most 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] Newborns / infants with genetic factors that make them prone to allergies and atopy are known to develop various allergic diseases such as infantile eczema, atopic dermatitis, food allergies, and even bronchial asthma and allergic rhinitis as they grow older (allergy march). Thus, since allergic diseases are likely to develop into another allergic disease once one disease has occurred and their treatment often takes a long time, it is necessary to suppress the onset of allergic diseases in infants.

[0004] As methods for detecting atopic dermatitis using biomarkers, detecting the peripheral blood eosinophil count, total serum IgE value, LDH (lactate dehydrogenase) value, Thymus and Activation-Regulated Chemokine (TARC) and Squamous cell carcinoma antigen 2 (SCCA2) in serum (Non-Patent Documents 1, 2, 3, etc.) have been proposed. Among them, it has been reported that TARC in serum reflects the disease state even in pediatric atopic dermatitis (Non-Patent Document 4). In the AD determination using serum TARC as an index in this report, it has been reported that the sensitivity and specificity of the determination are lower in children under 2 years old compared to children 2 years old and above.

[0005] 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 acids derived from living organisms can be extracted from bodily fluids such as blood, secretions, and tissues. 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 of the epidermis, sweat glands, hair follicles, and sebaceous glands can be detected from SSL (Patent Document 1). There is little concrete knowledge regarding the relationship between the expression of marker genes in SSL and the expression of marker genes in bodily fluids such as blood, and it is unclear whether disease markers in serum are expressed in SSL to a similar degree and can serve as markers with equivalent accuracy. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] International Public Gazette No. 2018 / 008319 [Non-patent literature]

[0007] [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 [Non-Patent Document 4] Fujisawa et al., Journal of the Japanese Society for Pediatric Allergy (2005) 19(5): 744-757 [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] The present invention relates to providing a method for detecting atopic dermatitis in infants and young children using lipids on the skin surface. [Means for solving the problem]

[0009] The inventors of this invention have found that the expression levels of TARC mRNA contained in SSL collected from infants with atopic dermatitis and infants with healthy skin without an allergic predisposition differ significantly between the two groups. They have discovered that this can be used as an indicator to detect atopic dermatitis in infants, and that it is particularly useful for detecting mild and moderate atopic dermatitis.

[0010] In other words, the present invention relates to the following 1) to 2). 1) A method for detecting atopic dermatitis in infants and young children, comprising the step of measuring the expression level of the TARC gene or its expression product in skin surface lipids collected from the subject. 2) A test kit for detecting atopic dermatitis in infants and young children, 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. [Effects of the Invention]

[0011] According to the present invention, it is possible to detect atopic dermatitis in infants and young children at an early stage with high accuracy, sensitivity, and specificity in a simple and non-invasive manner. [Brief explanation of the drawing]

[0012] [Figure 1] RNA expression levels of TARC in SSL in healthy children (HL) and infants with atopic dermatitis (AD). [Figure 2] RNA expression levels of TARC in SSL in healthy adults (HL) and adult atopic dermatitis patients (AD). [Figure 3] Serum TARC protein levels in healthy adults (HL) and adult atopic dermatitis patients (AD). [Modes for carrying out the invention]

[0013] All patent documents, non-patent documents, and other publications cited in this specification are hereby incorporated by reference in their entirety.

[0014] In the present invention, the terms "nucleic acid" or "polynucleotide" mean 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. are all included.

[0015] In the present invention, the "gene" includes double-stranded DNA containing human genomic DNA, single-stranded DNA (sense strand) containing cDNA, single-stranded DNA (complementary strand) having a sequence complementary to the sense strand, and fragments thereof, and means those in which some biological information is contained in the sequence information of the bases constituting the DNA. Further, the "gene" includes not only the "gene" represented by a specific base sequence, but also nucleic acids encoding homologs (i.e., homologs or orthologs) thereof, mutants such as gene polymorphisms, and derivatives. In the present invention, "TARC" is a kind of chemokine having leukocyte chemotactic activity, and refers to thymus and activation-regulated chemokine, a kind of chemokine produced by epidermal keratinocytes, dendritic cells, lymphocytes, vascular endothelial cells, etc., and is also called Chemokin ligand 17 (CCL17). The TARC gene (mRNA) is registered as the reference sequence number "NM_0029" of NCBI.

[0016] In the present invention, the "expression product" of a gene is a concept that includes the transcriptional product and translational product of the gene. The "transcriptional product" is RNA produced by transcription from a gene (DNA), and the "translational product" means a protein encoded by the gene that is translationally synthesized based on RNA. [[ID=2,3]]

[0017] In the present invention, "atopic dermatitis" refers to a disease mainly caused by itchy eczema that repeatedly worsens and remits, and many of its patients are considered to have an atopic predisposition. Examples of atopic predispositions include i) family history / past history (any one or more of bronchial asthma, allergic rhinitis / conjunctivitis, and atopic dermatitis), or ii) a predisposition to easily produce IgE antibodies.

[0018] In the present invention, "infant" in a broad sense refers to "child" before the onset of secondary sexual characteristics, specifically a concept including children under 12 years old, and preferably refers to infants from 0 years old until entering school age, specifically infants from 0 years old to 5 years old. The rash in atopic dermatitis that develops in infants starts from the head and face in the infancy stage and often descends to the trunk and extremities. In the toddler stage, the rash on the face decreases and appears mainly around the neck and joints of the extremities. Regarding the differences between infantile atopic dermatitis and adult atopic dermatitis, in recent years, it has been reported that adult atopic dermatitis shows epidermal keratinization abnormalities associated with chronic inflammatory abnormalities compared to infantile atopic dermatitis (Journal of Allergy and Clinical Immunology, Volume 141, Issue 6, June 2018, pages 2094 - 2106), but the number of reports is small and it is not clearly defined.

[0019] The degree (severity) of progression of atopic dermatitis is classified according to the state of the rash, for example, as asymptomatic, mild, mild (low severity), moderate (moderate severity), severe (high severity). Well-known 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.>), etc. For example, the EASI uses the head and neck, trunk, upper limbs, and lower limbs as evaluation sites, and calculates a value from 0 to 72 based on four symptom-specific scores for each evaluation site (erythema, edema / infiltration / papules, scratch marks, and lichenification) and the percentage (%) of the area of ​​these four symptoms in the total evaluation site. In the IGA, the evaluator (physician or researcher) comprehensively assesses the condition of the skin lesions on the whole body or a specific evaluation site and assigns a five-level rating: "no symptoms," "mild," "mild," "moderate," and "severe." Sometimes a six-level rating including "most severe" is used. IGA scores can be converted to an ordinal scale such as "no symptoms = 0," "mild = 1," "mild = 2," "moderate = 3," "severe = 4," and "most severe = 5."

[0020] In the present invention, "detection of atopic dermatitis in infants and young children" includes not only determining the presence (with or without symptoms) or absence (without symptoms) of atopic dermatitis in infants and young children, but also determining the degree of progression of atopic dermatitis in infants and young children, i.e., "mild," "moderate," and "severe." Preferably, this includes detecting "no symptoms," "mild," and "moderate" cases, respectively. Here, "mild" refers to a condition mainly characterized by dryness and mild erythema and scaling, corresponding to an EASI score greater than 0 but less than 6, or an IGA score of 2. "Moderate" refers to a condition mainly characterized by moderate erythema, scaling, a few papules, and scratch marks, corresponding to an EASI score of 6 or more but less than 23, or an IGA score of 3. "Severe" refers to a condition mainly characterized by severe swelling / edema / infiltration or lichenification accompanied by erythema, multiple papules, severe scaling, crusting, vesicles, erosions, numerous scratch marks, and prurigo nodules, corresponding to an EASI score of 23 or more but less than 72, or an IGA score of 4. "Asymptomatic" refers to a state of remission or near-remission with no symptoms, at the same level as a healthy child. Furthermore, in the assessment of "no symptoms," "mild," "moderate," and "severe" using EASI scores or IGA scores, the range of scores used as criteria for each assessment is not limited to the above and can be determined as appropriate.

[0021] In this invention, the term "detection" may be replaced with terms such as inspection, measurement, determination, evaluation, or evaluation support. However, in this specification, the terms "detection," "inspection," "determination," or "evaluation" do not include determination, evaluation, or diagnosis by a physician.

[0022] As shown in the examples described later, RNA expression analysis in SSL was performed on 20 healthy children and 16 children with atopic dermatitis (6 months to 5 years old), including 9 children diagnosed with mild (score greater than 0 but less than 6) and 7 children judged to have moderate (score between 6 and 23) according to the severity of EASI. The results showed that TARC gene expression increased in a severity-dependent manner. Furthermore, based on TARC RNA expression data derived from SSL in healthy children, mildly ill children, and moderately ill children, we verified the accuracy of distinguishing between healthy children and mildly to moderately ill children, healthy children and mildly ill children, and mildly ill children and moderately ill children. The accuracy rates ((true positive + true negative) / total) were 89%, 79%, and 87%, respectively, demonstrating that high accuracy in discrimination is possible. Furthermore, according to the method of the present invention, even when targeting infants under two years of age, which were considered difficult to distinguish using conventional methods, the accuracy of distinguishing between healthy children and children with mild to moderate symptoms, healthy children and children with mild symptoms, and children with mild symptoms and children with moderate symptoms is 75%, 70%, and 90%, respectively, indicating that all can be distinguished well.

[0023] Therefore, the TARC gene or its expression product in SSL is useful as an infant atopic dermatitis marker for detecting infant atopic dermatitis, and is particularly useful as a marker for distinguishing and detecting "no symptoms of infant atopic dermatitis," "mild or moderate infant atopic dermatitis," "mild infant atopic dermatitis," and "moderate infant atopic dermatitis." In this invention, the TARC gene (hereinafter also referred to as the "target gene") which can serve as a detection marker for atopic dermatitis in infants and young children includes genes having a base sequence substantially identical to the base sequence of the DNA constituting the TARC gene, insofar as it can serve as a biomarker for detecting atopic dermatitis in infants and young children. 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 DNA constituting the gene has 90% or more identity, preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more.

[0024] The present invention provides a method for detecting atopic dermatitis in infants and young children, which includes the step of measuring the expression level of the TARC gene or its expression product in an SSL sample taken from a subject.

[0025] In the present invention, the subjects from which SSL is collected are not particularly limited in terms of gender or race, as long as they are infants, but infants who require detection of atopic dermatitis or infants suspected of having atopic dermatitis are preferred. For example, such subjects include infants who have developed atopic dermatitis, infants with itchy eczema, infants judged to have an atopic predisposition based on family history and medical history, and infants who have a predisposition to easily produce IgE antibodies.

[0026] Here, "superficial 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, unless otherwise specified, "skin" is a general term for the region including the stratum corneum, epidermis, dermis, hair follicles, and tissues such as sweat glands, sebaceous glands, and other glands.

[0027] Any 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. The skin from which SSL is collected is not particularly limited and can be any part of the body, such as the head, face, neck, trunk, hands, or feet. Areas with high sebum secretion, such as the skin of the face, are preferred.

[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 target genes for measuring the expression level of the TARC gene or its expression product include 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, with RNA being preferred and mRNA being more preferred. Here, molecules that interact with RNA, DNA, or proteins include DNA, RNA, proteins, polysaccharides, oligosaccharides, monosaccharides, lipids, fatty acids, and their phosphorylated, alkylated, or 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 its expression product.

[0030] In the method of the present invention, in a preferred embodiment, the expression level of mRNA contained in SSL is analyzed, and preferably the RNA is converted to cDNA by reverse transcription, after which the cDNA or its amplified 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 or AGPC (acid guanidin RNA extraction can be performed using methods such as the ium thiocyanate-phenol-chloroform extraction method, or methods using columns such as TRIzol®, RNeasy®, or QIAzol®, methods using special magnetic particles coated with silica, 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, only TARC DNA may be amplified using a primer pair that targets the TARC DNA to be analyzed, or multiple DNA molecules, including TARC DNA, may be amplified simultaneously using multiple primer pairs. RT-PCR is an example of a method for amplifying only the DNA to be analyzed, while multiplex PCR is an example of a method for simultaneously amplifying multiple DNA molecules. Multiplex PCR is a method that simultaneously amplifies multiple gene regions by using multiple primer pairs 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 instance, the purified PCR reaction product can be prepared into a buffer solution, the amplified DNA can be subjected to removal of PCR primer sequences and adapter ligation, and the resulting reaction product can be amplified as needed to prepare a library for quantitative analysis. These operations can be performed, for example, using the 5×VILO RT Reaction Mix included with the SuperScript® VILO cDNA Synthesis kit (Life Technologies Japan Co., Ltd.), the 5×Ion AmpliSeq HiFi Mix included with the Ion AmpliSeq Transcriptome Human Gene Expression Kit (Life Technologies Japan Co., Ltd.), and the Ion AmpliSeq Transcriptome Human Gene Expression Core Panel, according to the protocols included with each kit.

[0036] When measuring the expression level of a target TARC 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 standard method. Subsequently, the double helix of the formed labeled DNA and RNA is measured by detecting the signal originating from the label.

[0037] When measuring the expression level of the target TARC 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 standard 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 TARC of the present invention are hybridized with it using this as a template. Then, PCR is performed according to a standard 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 TARC 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, and even more preferably 95% 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 TARC gene, which is the target of this invention, molecules that interact with said protein, molecules that interact with RNA, or molecules that interact with DNA, protein chip analysis, immunoassay (e.g., ELISA), mass spectrometry (e.g., LC-MS / MS, MALDI-TOF / MS), 1-hybrid method (PNAS 100, 12271-12276 (2003)), or 2-hybrid method (Biol. Reprod. 58, 302-311 (1998)), the methods used are protein chip analysis, immunoassay (e.g., ELISA), mass spectrometry (e.g., LC-MS / MS, MALDI-TOF / MS), 1-hybrid method (PNAS 100, 12271-12276 (2003)), and 2-hybrid method (Biol. Reprod. 58, 302-311 (1998)). Methods such as the above can be used and selected appropriately depending on the subject. 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 protein 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, a 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 TARC gene or its expression product in SSL collected from subjects is measured, and infantile atopic dermatitis is detected based on this expression level. Specifically, detection is performed by comparing the measured expression level of the TARC gene 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, which is the read count value corrected for the difference in the total number of reads between samples; the value obtained by converting the RPM value to a base-2 logarithm (Log2RPM value) or the base-2 logarithm with an integer 1 added (Log2(RPM+1) value); or the count value corrected using DESeq2 (Normalized count value) or the base-2 logarithm with an integer 1 added (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. Signal values ​​obtained by microarray methods and their corrected values ​​may also be used. Furthermore, when analyzing the expression level of only the target TARC gene using RT-PCR or similar methods, 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 (relative quantification) for analysis, or to quantify the absolute copy number using a plasmid containing the region of the target gene (absolute quantification) for analysis. The copy number obtained by digital PCR may also be used. Here, "control level" refers, for example, to the expression level of the TARC gene or its expression product in healthy children. The expression level in healthy children may be a statistical value (e.g., the mean) of the expression level of the TARC gene or its expression product measured from a healthy child population. Depending on the purpose of detection, the expression level of the TARC gene or its expression product in children with mild or moderate atopic dermatitis may also be used as the "control level".

[0043] Furthermore, in this invention, atopic dermatitis in infants and young children can also be detected by increasing or decreasing the expression level of the TARC gene or its expression product. In this case, the expression level of the TARC gene or its expression product in SSL derived from the subject is compared with the cutoff value (reference value) of the TARC gene or its expression product. The cutoff value can be determined using various statistical analysis methods. For example, values ​​based on ROC curve (Receiver Operatorating Characteristic curve) analysis (e.g., Youden's index, distance from the upper left corner coordinate (0,1) on the ROC curve, etc.) are examples. An ROC curve is created by plotting the following parameters: the vertical axis represents the probability of a positive result in a positive patient (True Position Fraction (TPF), sensitivity), and the horizontal axis represents the value obtained by subtracting the probability of a negative result in a negative patient (Specificity) from 1 (False Position Fraction (FPF)). The ROC curve is created by varying the cutoff point, which is the threshold value for determining whether a test result indicates a finding, as a mediating variable. The cutoff point to be adopted as the cutoff value from the created ROC curve should be determined based on the severity of the disease, the role of the test, and various other conditions. Typically, if the cutoff point is set at a point with a low false positive rate, the number of negative patients who test positive will decrease, but conversely, a large number of positive patients will be excluded, resulting in lower sensitivity. On the other hand, increasing sensitivity will increase the false positive rate among negative patients. Generally, to increase both sensitivity and specificity (to approach 1), the cutoff value is set to the value that gives the point closest to (0,1) on the ROC curve, or to the value (Youden index) that maximizes "true positive (sensitivity)" - "false positive (1 - specificity)".

[0044] The present invention provides a test kit for detecting atopic dermatitis in infants and young children, which contains a test reagent for measuring the expression level of the TARC gene or its expression product in silicic stromal cells (SSLs) isolated from patients. Specifically, this includes reagents for nucleic acid amplification and hybridization, such as oligonucleotides (e.g., primers for PCR) that specifically bind (hybridize) to the TARC gene or nucleic acids derived therefrom, or reagents for immunological measurement, such as antibodies that recognize the expression product (protein) of the TARC gene. The oligonucleotides, antibodies, etc., included in the kit can be obtained by known methods as described above. Furthermore, the test kit may also 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 to be used as positive and negative controls, tools for collecting SSL (e.g., oil-absorbing film for collecting SSL), reagents for storing the collected SSL, storage containers, and reagents for extracting and purifying RNA from the collected SSL.

[0045] Aspects and preferred embodiments of the present invention are shown below. <1> A method for detecting atopic dermatitis in infants and young children, comprising the step of measuring the expression level of the TARC gene or its expression product in skin surface lipids collected from the subject. <2> The expression level of a gene or its expression product is measured by the amount of mRNA expression. <1> The method. <3> The measured expression level is compared with the reference value of the gene or its expression product to evaluate the presence or absence of atopic dermatitis in infants and young children, or the degree of its progression. <1> or <2> The method. <4> The progression of atopic dermatitis in infants and young children is mild or moderate. <3> The method. <5> The degree of progression of atopic dermatitis in infants and young children is determined by the EASI score or the IGA score. <3> or <4> The method. <6> The subjects are infants and toddlers aged 0 to 5 years old. <1> ~ <4> One of the following methods. <7> The subjects are infants under 2 years old. <1> ~ <5> One of the following methods. <8> Use of TARC genes or their expression products derived from skin surface lipids collected from subjects as markers for atopic dermatitis in infants and young children. <9> The TARC gene or its expression product is mRNA contained in the skin surface lipids collected from the subject. <8> Use. <10> To assess the presence or absence of atopic dermatitis in infants and young children, or the degree of its progression. <8> or <9> Use. <11> The progression of atopic dermatitis in infants and young children is mild or moderate. <10> Use as described. <12> The degree of progression of atopic dermatitis in infants and young children is determined by the EASI score or the IGA score. <10> or <11> Use as described. <13> The subjects are infants and toddlers aged 0 to 5 years old. <8> ~ <12> Use of either of the following. <14> The subjects are infants under 2 years old. <8> ~ <13> Use of either of the following. <15> 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> ~ <7> A test kit for detecting atopic dermatitis in infants and young children, used in one of the following methods. [Examples]

[0046] The present invention will be described in more detail below based on examples, but the present invention is not limited thereto.

[0047] Example 1: Detection of the TARC gene in RNA extracted from SSL 1) SSL collection The study included 20 healthy-skinned infants (HL) (6 months to 5 years old, male and female) and 16 infants with atopic dermatitis (AD) (6 months to 5 years old, male and female). The infants 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 infants with a score greater than 0 but less than 6 were classified as mild, while those with a score of 6 or greater but less than 23 were classified as moderate (Chopra et al. Br J Dermatol. 177, 2017). The results showed that, Nine subjects had mild symptoms, and seven had moderate symptoms. Of the subjects, 14 had HL and 10 had AD (6 mild and 4 moderate) under the age of two. Sebum was collected from the entire face of each subject (including the affected area in the case of AD) using an oil-absorbing film (5 x 8 cm, made of polypropylene, 3M). The oil-absorbing film was then transferred to a vial and stored at -80°C for approximately one month until it was ready for RNA extraction.

[0048] 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.).

[0049] 3) Analysis of TARC gene expression RNA expression data (read count values) extracted from SSLs of healthy, mildly affected, and moderately affected infants, as measured in 2) above, were obtained, and the data were normalized and expression variation analysis was performed using DESeq2. Genes with a corrected p-value (FDR) of less than 0.05 in AD compared to healthy individuals were selected, and the result showed that TARC gene expression was significantly higher in AD (FDR < 0.05), indicating that TARC is useful as a detection marker for infant atopic dermatitis in SSLs, similar to that in serum. Differences in expression levels are shown based on the base-2 logarithm (Log2(Normalized count + 1) value) obtained by adding the integer 1 to the normalized count value. (Figure 1).

[0050] 4) Verification of discrimination accuracy Based on the TARC RNA expression data (Log2(Normalized count+1) values) from healthy children, mildly ill children, and moderately ill children used in 3) above, the ROC curve was calculated. A line was created, and the cutoff value was set to the value (Youden index) at which "true positive (sensitivity)" - "false positive (1 - specificity)" is maximized. When the cutoff values ​​for distinguishing between healthy children and children with mild to moderate symptoms, healthy children and children with mild symptoms, and children with mild symptoms and children with moderate symptoms were set to 13.5, 12.5, and 13.8, respectively, as shown in Tables 1a to 1c below, accurate detection rates of 89%, 79%, and 87% were achieved, demonstrating that detection is possible with superior accuracy (sensitivity and specificity) compared to conventional methods.

[0051] [Table 1]

[0052] Furthermore, the results of a similar verification conducted with the target group limited to infants under 2 years of age are shown in Tables 2a-2c below. The value at which "True positive (sensitivity)" - "False positive (1 - specificity)" is maximized (Youden By setting the index, and setting cutoff values ​​within the numerical ranges of 12.5-12.8, 12.5-12.8, and 13.6-13.8 to distinguish between healthy children and children with mild to moderate symptoms, healthy children and children with mild symptoms, and children with mild to moderate symptoms, respectively, accurate determinations were achieved with accuracy rates of 75%, 70%, and 90%, as shown in Tables 2a to 2c below. This demonstrated that the method is useful even for infants under 2 years old, where conventional methods were more difficult.

[0053] [Table 2]

[0054] The following example illustrates, for reference, that serum TARC levels and TARC expression levels in SSL-derived RNA do not necessarily correlate, using the case of an adult patient with atopic dermatitis.

[0055] Reference example: Detection of TRAC in adult-derived samples (SSL, serum) 1) SSL collection The study included 14 healthy adult subjects (HL) (25-57 years old, male) and 29 adult subjects with atopic dermatitis (AD) (23-56 years old, male). Subjects with atopic dermatitis had been diagnosed by a dermatologist as having mild or moderate atopic dermatitis. Sebum was collected from each subject's entire face using an oil-absorbing film (5 x 8 cm, polypropylene, 3M), and the oil-absorbing film was transferred to a vial and stored at -80°C for approximately one month until use for RNA extraction. 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.). 3) Analysis of TARC gene expression RNA expression data (read count values) extracted from SSLs of healthy and AD patients measured in 2) above were obtained, and the data were normalized and differential expression analysis was performed using DESeq2. Genes with a corrected p-value (FDR) of less than 0.05 in the likelihood ratio test compared to healthy individuals were selected. As a result, the TARC gene was not selected as a differentially expressed gene, indicating that TARC did not show a significant difference in expression levels in RNA derived from SSLs of adult AD patients compared to healthy individuals. The base-2 logarithm (Log2(Normalized count value)) was obtained by adding the integer 1 to the normalized count value. Figure 2 shows the differences in expression levels based on the ount+1) value. 4) Blood collection 1) For subjects from whom SSL was collected (14 HLs and 29 ADs), a physician or nurse collected blood in blood collection tubes (Venoject® II-P, TERUMO) according to standard procedures. The blood collection tubes were centrifuged at room temperature (3000 rpm, 15 minutes) and stored refrigerated until analysis. 5) Analysis of serum TARC levels Serum TARC levels were analyzed by an external laboratory (LSI Medience Corporation) using CLEIA (chemiluminescent enzyme immunoassay). A comparison of serum TARC protein levels between healthy individuals and AD patients revealed significantly higher levels in AD patients (unpaired t-test, p=0.014) (Figure 3).

Claims

1. A method for measuring the expression level of mRNA of the TARC gene in a subject, comprising a step of measuring the expression level of mRNA of the TARC gene in lipids on the skin surface collected from the entire face of the subject using a sheet-shaped lipid-absorbent material on the skin surface in order to detect the degree of progression of infant atopic dermatitis in the subject.

2. The method of claim 1, wherein the detection comprises comparing a measured value of the expression level with a reference value of the expression level of mRNA of the gene to assess the degree of progression of infantile atopic dermatitis.

3. 3. The method according to claim 2, wherein the degree of progression of infant atopic dermatitis is mild or moderate.

4. The method according to any one of claims 1 to 3, wherein the subject is an infant aged 0 to 5 years.

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