Method for detecting severity of infantile facial eczema

Abstract

To provide a detection marker for detecting the severity of infantile facial eczema and a method for detecting the severity of infantile facial eczema using the detection marker.SOLUTION: A method for detecting the severity of infantile facial eczema includes a step for measuring, in a biological sample collected from an infantile test subject, the expression level of at least one gene selected from the group of eight genes consisting of BABAM1, PDLIM5, PMS2P5, SNHG8, COG5, GPR132, PSMD7, and CCDC134 or an expression product thereof.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for detecting the severity of infant facial eczema using a severity detection marker for infant facial eczema.

Background Art

[0002] Eczema (dermatitis) is a general term for inflammation that occurs on the surface of the skin. As infants grow, they develop various types of eczema (dermatitis). Representative ones include neonatal acne, seborrheic eczema, contact dermatitis (rash), atopic dermatitis (AD), etc. Atopic dermatitis is a disease mainly characterized by itchy eczema that反复 exacerbates and remits, and many patients have an atopic predisposition (family history, past history, or a predisposition to produce IgE antibodies) (Non-Patent Document 1). Neonatal acne is a rash similar to acne mainly seen on the face around 2 weeks after birth and improves transiently. Seborrheic eczema is an eczema that causes erythema with scaling in seborrheic areas (the head and face in infants). In infants, it is seen around 1 month after birth and often improves spontaneously thereafter. Contact dermatitis includes irritant contact dermatitis, which occurs when the skin comes into contact with some substance and that substance causes inflammation, and allergic contact dermatitis, which occurs at the site where an antigen contacts an individual sensitized to that antigen, and is an eczema that causes erythema, edema, papules, blisters, etc. Contact dermatitis is caused by contact with a causative substance that causes irritation, but improves by removing it.

[0003] Such various types of infantile eczema are also suggestive of future impacts. In atopic dermatitis, it has been reported that it is related to the subsequent allergy march (successively developing allergies starting from one allergic onset), and its prevention, early detection, and treatment intervention are considered important. Also, in other types of eczema, if the state of inappropriate treatment continues, it may lead to the development of allergic diseases including atopic dermatitis due to a decrease in the skin barrier function, invasion of allergens, and establishment of sensitization due to long-term exposure to allergens. Therefore, similar to atopic dermatitis, its prevention, early detection, and treatment intervention are important (Non-Patent Document 2).

[0004] While a dermatologist's diagnosis is crucial for understanding the state of eczema, the decision to seek medical attention is left to the discretion of the parents, and since their criteria for judgment differ from person to person, there is a risk of missing the opportunity for early and appropriate treatment. Therefore, a technology that can more easily assess the state of eczema is expected to lead to appropriate skincare for infants and young children and support their healthy growth.

[0005] In recent years, technologies have been developed to investigate the current and future physiological state of the human body by analyzing nucleic acids such as DNA and RNA in biological samples. Nucleic acids derived from living organisms can be extracted from bodily fluids such as blood, secretions, and tissues. More recently, it has been reported that RNA contained in skin surface lipids (SSL) can be used as a sample for biological analysis (Patent Document 1). [Prior art documents] [Patent Documents]

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

[0007] [Non-Patent Document 1] J Allenrgy Clin Immunol Pract.2020;8:1721-1724 [Non-Patent Document 2] J Allergy Clin Immunol.2008;121:1331-6 [Overview of the project] [Problems that the invention aims to solve]

[0008] The present invention relates to a detection marker for detecting the severity of facial eczema in infants and young children, and a method for detecting the severity of facial eczema in infants and young children using the detection marker. [Means for solving the problem]

[0009] The inventors collected SSL from the entire face of infants with facial eczema, excluding the eye and mouth areas, and comprehensively analyzed the RNA expression status contained in SSL as sequencing information. As a result, they found that the expression level of specific genes significantly correlated with the severity of infant facial eczema, and that this could be used as an indicator to detect the severity of infant facial eczema.

[0010] The present invention relates to the following 1) to 3). 1) A method for detecting the severity of facial eczema in an infant, comprising the step of measuring the expression level of at least one gene selected from eight gene groups, namely BABAM1, PDLIM5, PMS2P5, SNHG8, COG5, GPR132, PSMD7, and CCDC134, or its expression product, in a biological sample taken from the infant. 2) A test kit for detecting the severity of infantile facial eczema, used in the method of 1), comprising an oligonucleotide that specifically hybridizes with the gene or nucleic acid derived therefrom, or an antibody that recognizes the expression product of the gene. 3) A detection marker for detecting the severity of infantile facial eczema, comprising at least one gene selected from the eight gene groups BABAM1, PDLIM5, PMS2P5, SNHG8, COG5, GPR132, PSMD7, and CCDC134, or its expression product. [Effects of the Invention]

[0011] According to the present invention, it is possible to detect the severity of facial eczema in infants and young children in a simple and non-invasive manner. This allows parents to understand the condition of their infant's facial eczema based on objective indicators, and to take appropriate measures against the condition. [Brief explanation of the drawing]

[0012] [Figure 1] A graph showing the relationship between the predicted facial eczema score obtained by the facial eczema score prediction model and the diagnostic score. [Modes for carrying out the invention]

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

[0014] In this 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.

[0015] 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 this invention includes not only "genes" represented by a specific base sequence, but also their homologs (i.e., homologs or orthologs), variants such as gene polymorphisms, and derivatives. Herein, the gene names disclosed herein follow the Official Symbols listed in NCBI ([www.ncbi.nlm.nih.gov / ]).

[0016] 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.

[0017] In the present invention, "facial eczema" refers to inflammation that occurs on the skin of the face. Clinically, erythema, papules, maceration, scaling, and scratching marks can be seen. When the symptoms become severe, the erythema becomes erythema with edema, the papules have pustules, and the maceration changes to erosion. Sites where facial eczema is likely to occur include the forehead, lateral head, lower eyelid, zygomatic bone, parotid masseter, cheek, chin, etc. "Infant" in a broad sense is a concept that includes "children" before the onset of secondary sexual characteristics, specifically children under 12 years old, and preferably refers to infants from 0 years old until they enter school age, specifically infants from 0 years old to 5 years old.

[0018] The "severity" of facial eczema refers to the degree of severity of the symptoms of facial eczema. For example, it is classified as no symptoms, mild, mild, mild to moderate, moderate, moderate to severe, severe. The severity of facial eczema can be determined based on, for example, a known evaluation score (hereinafter also referred to as the "facial eczema score") for evaluating the symptoms of facial eczema. Examples of the facial eczema score include giving a 7-point score of 0: no symptoms, 1: mild, 2: mild, 3: mild to moderate, 4: moderate, 5: moderate to severe, 6: severe for 5 symptoms including erythema, papules, maceration, scaling, and the scratching marks corresponding to crusting from the eczema triangle (indicating the state of the eczema reaction from onset), and the sum of these scores (total value) (see Pediatric Dermatology (2014) Vol. 31 No. 1 p.1-7, and Dermatology (2000) 200 p.238-243). Specifically, the facial eczema score is 0: no symptoms, 1-5: mild, 6-10: mild, 11-15: mild to moderate, 16-20: moderate, 21-25: moderate to severe, 26-30: severe. In the present invention, the facial eczema score itself may also be used as an index indicating the severity of infantile facial eczema.

[0019] In the present invention, the "detection" of the severity of infantile facial eczema can also be rephrased using terms such as examination, measurement, determination, or evaluation support. Note that the terms "detection", "examination", "measurement", "determination", or "evaluation" of the severity of infantile facial eczema in the present invention do not include the diagnosis of the severity of infantile facial eczema by a doctor.

[0020] As shown in the examples described below, SSL was collected from the entire face of infants with facial eczema, excluding the eyes and mouth, and the expression level data of RNA derived from the SSL was obtained by the following procedures 1) to 3), and 10 genes shown in Table 1 were selected as feature genes. And it was shown that the severity of infantile facial eczema can be detected by a discriminant formula (prediction model of facial eczema score) using the feature genes. Therefore, a gene selected from the gene group shown in Table 1 or its expression product can be used as a detection marker, and the severity of infantile facial eczema can be detected based on its expression level. 1) Obtain data (read count values) of the expression level of RNA derived from SSL. 2) Convert the read count value to an RPM value corrected for the difference in the total number of reads among the sample test infants, add the integer 1 to the RPM value, and convert it to a base-2 logarithm value (log2(RPM + 1) value), and calculate the Spearman's rank correlation coefficient for the facial eczema score. 3) Select genes with a large Spearman's rank correlation coefficient (ρ) (top genes) as feature genes.

[0021]

Table 1

[0022] Among the 10 genes, 8 genes, BABAM1, PDLIM5, PMS2P5, SNHG8, COG5, GPR132, PSMD7, and CCDC134, which are marked with * in the table and shown in bold, are genes whose relationship with infantile facial eczema has not been reported so far. A gene selected from these gene groups or its expression product is a novel detection marker for detecting the severity of infantile facial eczema. In this invention, at least one gene selected from the eight gene groups or its expression product is used as a detection marker, and the severity of infantile facial eczema is detected based on its expression level. Furthermore, in addition to at least one gene selected from the eight gene groups or its expression product, it is also possible to use at least one gene selected from the two gene groups NRIP1 and PPIA or its expression product as a detection marker, and detect the severity of infantile facial eczema based on its expression level. By detecting the severity of facial eczema in infants and young children, it is possible to understand the condition of the eczema in infants and young children, such as whether or not eczema has developed, the progression of the eczema, the degree of healing of the eczema, and the effectiveness of treatment or preventive measures against the eczema.

[0023] The eight genes BABAM1, PDLIM5, PMS2P5, SNHG8, COG5, GPR132, PSMD7, and CCDC134 can each individually serve as detection markers for detecting the severity of infantile facial eczema. Of these, preferably two or more, more preferably five or more, and even more preferably all eight, are used in combination. In particular, it is preferable to select two or more that include BABAM1 as an essential gene. Furthermore, the at least one selected from NRIP1 and PPIA to be combined with the eight genes is preferably PPIA, and it is more preferable to select two or more from the eight genes, NRIP1 and PPIA, including BABAM1 and PPIA, and even more preferable to select all 10 genes shown in Table 1.

[0024] The genes that can serve as detection markers for detecting the severity of infantile facial eczema (hereinafter also referred to as "target genes") include genes that have substantially identical base sequences to the DNA that constitutes the target gene, insofar as they can serve as biomarkers for detecting the severity of infantile facial eczema. 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 target gene has 90% or more identity, preferably 95% or more, more preferably 98% or more identity, and even more preferably 99% or more identity.

[0025] The present invention provides a method for detecting the severity of facial eczema in infants, comprising the step of measuring the expression level of a target gene, in one embodiment, at least one gene selected from a group of eight genes: BABAM1, PDLIM5, PMS2P5, SNHG8, COG5, GPR132, PSMD7, and CCDC134, or its expression product, in a biological sample taken from a test infant. In another embodiment, the method includes measuring the expression level of at least one gene or its expression product selected from the eight gene groups, and the expression level of at least one gene or its expression product selected from the two gene groups NRIP1 and PPIA, in a biological sample taken from a test infant.

[0026] In this invention, the subjects of the test include, for example, infants who desire or need the detection of infantile facial eczema. For example, the subjects of the test include infants who have developed infantile facial eczema, infants suspected of having developed infantile facial eczema, infants with a genetic predisposition to infantile facial eczema, or infants whose close relatives, such as siblings, have developed or have developed infantile facial eczema.

[0027] The biological samples used in the present invention may be any cells, tissues, and biomaterials in which the gene expression of the present invention is altered. 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, feces, hair, etc. Preferably, skin or surface lipids (SSL), more preferably surface lipids (SSL). The area of ​​skin from which SSL is collected is not particularly limited and may include skin from any part of the body such as the head, face, neck, trunk, hands and feet. Preferably, areas prone to infantile facial eczema include the forehead, temporal region, infraorbital region, zygomatic region, parotid gland masseter muscle region, cheek region, chin region, or the entire face including these areas. The area of ​​skin from which SSL is collected may be either a lesion area where infantile facial eczema is present or an unaffected area, but preferably a lesion area or an unaffected area near a lesion area. Here, "near a lesion area" refers to an area within 10 cm adjacent to the lesion area.

[0028] 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 the present invention, 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.

[0029] Any means used for the collection or removal of SSL from the skin can be employed to collect SSL from the skin of the test infant. 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 or scraper. 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.

[0030] RNA-containing SSLs collected from test infants 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] In PCR, a primer pair targeting a specific DNA to be analyzed may be used to amplify only that specific DNA, or multiple primer pairs may be used to 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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).

[0042] 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 a 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 preferable to have 80% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 98% or more of identity in the base sequence. The identity of the base sequence 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 naturally occurring. Furthermore, the probes used for hybridization are usually labeled.

[0043] 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 method is carried out by contacting a biological sample with an antibody that specifically recognizes the expression product of the present invention, specifically an antibody that recognizes a structural characteristic site (epitope) that can distinguish the expression product protein from other proteins, detecting the polypeptide or protein in the sample 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, fluorescent substance, or enzyme, and the signal derived from these labeling substances is measured with 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)).

[0044] Thus, the expression level of the target gene of the present invention or its expression product in a biological sample taken from the infant is measured, and the severity of the facial eczema in the infant is detected based on the expression level. Furthermore, in this invention, the expression level of the target gene or its expression product in a biological sample taken from a test infant can be measured at least twice, and the presence or absence of a change in the severity of facial eczema in the test infant, or the degree of change, can be detected using the change in expression level or the amount of change as an indicator. Specifically, detection is performed by comparing the measured expression level of the target gene or its expression product of the present invention with a predetermined cutoff value (reference value). 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(Normalized count+1) value). Alternatively, values ​​calculated using common RNA-seq quantitative methods such as fragments per kilobase of exon per million reads mapped (FPKM), reads per kilobase of exon per million reads mapped (RPKM), transcripts per million (TPM), etc., may also 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 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 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.

[0045] Here, the "cutoff value" ("reference value") can be predetermined based on the relationship between the facial eczema score and the expression level of the target gene or its expression product of the present invention. For example, a certain population can be divided into groups based on the state of facial eczema, i.e., the facial eczema score, into groups of no symptoms, mild, mild, mild to moderate, moderate, moderate to severe, and severe. A value determined by referring to statistical values ​​such as the mean and standard deviation of the expression level of the target gene or its expression product in each group can then be determined as the cutoff value (reference value) for determining whether or not a person belongs to each group. When using multiple genes as target genes, it is preferable to determine a cutoff value (reference value) for each gene or its expression product. Groups may be formed based on gender, race, or age.

[0046] Furthermore, by utilizing the measured expression levels of the target gene or its expression product of the present invention, a discriminant formula (predictive model) can be constructed, and the severity of infantile facial eczema can be detected using this discriminant formula. For example, by using machine learning with the measured expression levels of the target gene or its expression product as the explanatory variable and the facial eczema score (for example, a value obtained by converting the score to a standard score) as the dependent variable, an optimal discriminant formula (predictive model) for detecting the severity of infantile facial eczema can be constructed. Then, the expression level of the target gene or its expression product of the present invention is similarly measured from biological samples taken from the test infants, and the obtained measurement values ​​are input into the discriminant formula (predictive model). The result obtained from the discriminant formula (predicted value of facial eczema score) can be detected as the severity of facial eczema in the test infants.

[0047] Furthermore, by utilizing the measured expression levels of target genes or their expression products derived from infants with facial eczema of different severity levels and those derived from healthy infants, a discriminant formula (predictive model) can be constructed to separate infants with facial eczema of different severity levels (for example, two or more groups selected from severity levels such as mild, mild, mild to moderate, moderate, moderate to severe, severe, etc.) from healthy infants (asymptomatic). This discriminant formula can then be used to detect the severity of facial eczema in infants. Specifically, using the expression levels of target genes or their expression products from infants with facial eczema of different severity levels and the measured expression levels of target genes or their expression products from healthy infants as training samples, a discriminant formula (predictive model) is constructed to separate infants with facial eczema of different severity levels (for example, two or more groups selected from severity levels such as mild, mild, mild to moderate, moderate, moderate to severe, severe, etc.) and healthy infants (no symptoms). Based on this discriminant formula, a cutoff value (reference value) is determined to distinguish each group of infants with facial eczema of different severity levels. Then, by similarly measuring the level of the target gene or its expression product from a biological sample taken from the infant, substituting the obtained measurement value into the discriminant formula, and comparing the result obtained from the discriminant formula with a cutoff value (reference value), the severity of facial eczema in the infant can be detected.

[0048] For constructing the discriminant (predictive model), publicly known algorithms such as those used in machine learning can be utilized. Examples of machine learning algorithms include Random Forest, Support Vector Machine (SVM linear), Support Vector Machine (SVM rbf), Neural Network, Generalized Linear Model, Regularized Linear Discriminant Analysis, Regularized Logistic Regression, and Lasso (Least Absolute Shrinkage and Selection Operator) Regression. By inputting validation data into the constructed predictive model and calculating predicted values, the model whose predicted values ​​best match the observed values, for example, the model with the highest accuracy, can be selected as the optimal predictive 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 predictive model. Furthermore, the root mean square error (RMSE) between the predicted and actual values ​​can be used as an accuracy evaluation metric for the prediction model, and the model with the smallest RMSE can be selected as the optimal prediction model.

[0049] 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 formula (predictive model). In an ROC curve, the vertical axis plots the probability of a positive result in a positive subject (sensitivity), and the horizontal axis plots the value obtained by subtracting the probability of a negative result in a negative subject (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).

[0050] The present invention provides a test kit for detecting the severity of infantile facial eczema, 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 test infant. 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).

[0051] With regard to the embodiments described above, the present invention further discloses the following aspects.

[0052] <1> A method for detecting the severity of facial eczema in an infant, comprising the step of measuring the expression level of at least one gene or its expression product selected from eight gene groups: BABAM1, PDLIM5, PMS2P5, SNHG8, COG5, GPR132, PSMD7, and CCDC134, in a biological sample taken from the infant.

[0053] <2> The expression levels of any of the eight genes selected from the group of eight genes BABAM1, PDLIM5, PMS2P5, SNHG8, COG5, GPR132, PSMD7, and CCDC134 are measured. Preferably, two or more genes, more preferably five or more genes, even more preferably two or more genes including BABAM1 as an essential gene, and even more preferably all eight genes or their expression products are measured. <1> The detection method described. <3> Preferably, the process further includes measuring the expression level of at least one gene selected from two gene groups, NRIP1 and PPIA, or its expression product. <1> or <2> The detection method described. <4> Preferably, the expression levels of PPIA, more preferably two or more genes including BABAM1 and PPIA, and even more preferably all 10 genes or their expression products shown in Table 1 are measured. <3> The detection method described. <5> The biological sample is preferably a bodily fluid from the infant or child being tested, such as organs, skin, blood, urine, saliva, sweat, stratum corneum, surface lipids (SSL), tissue exudate, serum, plasma, feces, or hair prepared from blood, more preferably skin or surface lipids (SSL), and even more preferably surface lipids (SSL). <1> ~ <4> A detection method described in any of the following. <6> The target for measuring the expression level of the gene or its expression product is preferably a cDNA artificially synthesized from RNA, the DNA encoding that RNA, the protein encoded by that RNA, a molecule that interacts with that protein, a molecule that interacts with that RNA, or a molecule that interacts with that DNA. <1> ~ <5> A detection method described in any of the following. <7> The expression level of the gene or its expression product is preferably the expression level of mRNA collected from the skin surface lipids (SSL) of the infant being tested. <1> ~ <5> A detection method described in any of the following. <8> The aforementioned infants or toddlers are those who have developed infantile facial eczema, those suspected of developing infantile facial eczema, those with a genetic predisposition to infantile facial eczema, or those whose close relatives, such as siblings, have developed or previously developed infantile facial eczema. <7> The detection method described. <9> The skin of the infant subject is from the forehead, temporal region, infraorbital region, zygomatic region, parotid masseter muscle region, cheek region, or chin region. <7> or <8> The detection method described. <10> The skin site from which surface lipids (SSL) are collected may be either a lesional area where infantile facial eczema is present or a non-lesion area, but it is preferably a lesional area or a non-lesion area near a lesional area. <7> ~ <9> A detection method described in any of the following. <11> Does not include facial eczema caused by atopic dermatitis. <1> ~ <10> A detection method described in any of the following. <12> This includes detecting the severity of facial eczema in the infant based on the expression level of the gene or its expression product. <1> ~ <11> A detection method described in any of the following. <13> Preferably, the method includes detecting the severity of infantile facial eczema using a discriminant formula (predictive model) based on the expression level of the gene or its expression product. The aforementioned discriminant formula (predictive model) is constructed by machine learning, with the measured expression level of the gene or its expression product as the explanatory variable and the facial eczema score as the dependent variable. <1> ~ <12> A detection method described in any of the following. <14> The severity of the aforementioned infantile facial eczema is determined by assigning a score to each of the five symptoms of the evaluated area—erythema, papules, maceration, desquamation, and scratch marks—on a 7-point scale: 0: no symptoms, 1: mild, 2: mild, 3: mild to moderate, 4: moderate, 5: moderate to severe, and 6: severe. The severity corresponds to the sum of these scores. <1> ~ <13> A detection method described in any of the following. <15> The evaluation area is the entire face of the infant. <14> The detection method described. <16> 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> ~ <15> A test kit used to detect the severity of facial eczema in infants and young children, which is used in the detection method for [the condition]. <17> A detection marker for detecting the severity of infantile facial eczema, comprising at least one gene selected from the eight gene groups BABAM1, PDLIM5, PMS2P5, SNHG8, COG5, GPR132, PSMD7, and CCDC134, or its expression product. <18> Use of at least one gene or its expression product, selected from eight gene groups—BABAM1, PDLIM5, PMS2P5, SNHG8, COG5, GPR132, PSMD7, and CCDC134—derived from biological samples taken from test infants, as a detection marker for the severity of infantile facial eczema. <19> The gene or its expression product is mRNA contained in the surface lipids of the skin of the infant being tested. <18> Use as described. [Examples]

[0054] Example 1: Detection of facial eczema severity using RNA extracted from SSL 1) Measurement of the subject infant and facial eczema score Sixty-one Japanese infants, both male and female, aged 3 to 8 months were included as test subjects. A dermatologist confirmed that none of the test subjects had been diagnosed with atopic dermatitis. For the entire face of the infants and toddlers being examined, a dermatologist assessed each of the five symptoms—erythema, papules, maceration, desquamation, and scratch marks—based on the criteria shown in Table 2, and assigned a score to each. The sum of these scores (total value) was then used as the facial eczema score. The results showed that there was 1 patient with no symptoms (score 0), 47 with mild symptoms (score 1-5), 11 with mild symptoms (score 6-10), 2 with mild to moderate symptoms (score 11-15), 0 with moderate symptoms (score 16-20), 0 with moderate to severe symptoms (score 21-25), and 0 with severe symptoms (score 26-30).

[0055] [Table 2]

[0056] 2) SSL collection Sebum was collected from the entire face of each test infant, excluding the eye and mouth areas, 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 until used for RNA extraction.

[0057] 3) RNA preparation and sequencing The oil-absorbing film described in 2) 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.), and then the buffer was reconstituted, primer sequences were digested, adapter ligation and purification were performed, and amplification was carried out 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.).

[0058] 4) Data Analysis (1) Data used The expression levels (read counts) of SSL-derived RNA measured in step 3) above were obtained and converted to RPM values ​​corrected for differences in the total number of reads among the sample infants. To approximate the RPM values, which follow a negative binomial distribution, to a normal distribution, the base-2 logarithm (log2(RPM+1) value) obtained by adding an integer 1 to the RPM value was used. Of the expression level data from all sample infants, only 5100 genes for which expression level data without missing values ​​was obtained for more than 90% of the sample infants were used for the following analysis.

[0059] (2) Dataset splitting Of the 61 infants and toddlers in the study, RNA profile data from 50 were used as training data for model construction, and RNA profile data from the remaining 11 were used as test data for evaluating model accuracy. Data splitting was performed using the [R] createDataPartition function, ensuring that the distribution of the target variable was uniform between the training and test datasets.

[0060] (3) Selection of characteristic genes Using the train data partitioned in (2), feature genes that were predicted to be suitable for the classification prediction model were selected based on Spearman's correlation coefficient. Spearman's rank correlation coefficient was calculated for each combination of facial eczema scores from the 50 infant subjects (the source of the train data) and all gene RNA expression data. As a result, the top 10 genes with the largest Spearman rank correlation coefficient (ρ), as shown in Table 3, were selected as characteristic genes. The eight genes marked with an asterisk (*) and shown in bold in the table were genes that had not previously been reported to be associated with infantile facial eczema.

[0061] [Table 3]

[0062] (4) Creating a facial eczema score prediction model using the caret package The top 10 genes with a high correlation to facial eczema scores were selected as features, and a facial eczema score prediction model was constructed using the caret package. The expression levels (log2(RPM+1) values) of the aforementioned feature genes selected from SSL-derived RNA were used as explanatory variables, and the facial eczema score converted to a standard score was used as the dependent variable. Using the features selected above, facial eczema score prediction models were constructed on the training data using six algorithms from the caret package: linear regression (Lm), random forest (Rf), neural network (Nnet), Lasso regression (Lasso), rbf kernel support vector machine (SVM rbf), and linear kernel support vector machine (SVM linear). To mitigate the impact of data bias on the models, the models were cross-validated 10 times. The RMSE (Root Mean Squared Error) was calculated as an indicator of the best predictive model, and the model with the smallest value was selected as the best model. Next, the gene expression levels (log2(RPM+1) values) of the features from the test data were input to the best model, and the predicted facial eczema score (predicted score) was calculated. The PEARSON correlation coefficient between the obtained predicted score and the actual diagnostic score was calculated, and a value closer to 1 indicated a model with higher prediction accuracy.

[0063] (5) Results As shown in Figure 1, when the top 10 genes with the highest correlation coefficients to the facial eczema score were used as features, the random forest model (Model 1) achieved the highest accuracy with an RMSE of 9.61. The correlation coefficient between the predicted score and the diagnostic score using the test data at that time was 0.718 (p=0.019). This revealed that the 10 genes extracted based on their correlation with the facial eczema score are important genes for predicting the regression of the infant facial eczema score and are useful for detecting the severity of infant facial eczema. Furthermore, when a facial eczema score prediction model was constructed using two genes, BABAM1 and PPIA, selected from 10 types, the linear regression model showed the highest accuracy with an RMSE of 10.61. The correlation coefficient between the predicted score and the diagnostic score based on the test data was 0.575 (p=0.064), confirming that facial eczema scores can be predicted. Furthermore, when a facial eczema score prediction model was constructed using only one of either BABAM1 or PPIA, the correlation coefficients between the predicted score from the test data and the diagnostic score were 0.545 (neural network, RMSE=11.02) (p=0.08) and 0.619 (linear regression model, RMSE=10.02) (p<0.05), respectively, revealing that these are important genes.

Claims

1. A method for detecting the severity of facial eczema in a test infant, comprising a step of measuring the expression level of at least one gene or its expression product selected from a group of eight genes, namely BABAM1, PDLIM5, PMS2P5, SNHG8, COG5, GPR132, PSMD7 and CCDC134, in skin surface lipids collected from the skin of the test infant.

2. The method for detection according to claim 1, further comprising a step of measuring the expression level of at least one gene selected from the two gene groups of NRIP1 and PPIA or its expression product.

3. The method according to claim 1, wherein the expression level of the gene or its expression product is the expression amount of mRNA.

4. 2. The method according to claim 1, wherein the skin of the subject infant is skin of the forehead, temporal region, infraorbital region, zygomatic region, parotid gland masseter muscle region, cheek or chin region.

5. The method for detection according to claim 1, further comprising detecting the severity of facial eczema in the subject infant based on the expression level of the gene or its expression product.

6. A test kit for detecting the severity of infant facial eczema, which is used in the detection methods of claims 1 to 5, and which contains an oligonucleotide that specifically hybridizes with the gene or a nucleic acid derived therefrom, or an antibody that recognizes the expression product of the gene.

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