Method for detecting severity of infantile diaper dermatitis

Abstract

To provide a detection marker for detecting the severity of infantile diaper dermatitis and a method for detecting the severity of infantile diaper dermatitis using the detection marker.SOLUTION: A method for detecting the severity of infantile diaper dermatitis 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 four genes consisting of GALNT3, CMTM6, SLC35E1, and EID3 or an expression product thereof.SELECTED DRAWING: None

Description

Technical field

[0001] The present invention relates to providing a method for detecting the severity of infant diaper dermatitis using an infant diaper dermatitis severity detection marker. [Background technology]

[0002] Eczema (dermatitis) is a general term for inflammation that occurs on the surface of the skin. Infants develop various types of eczema (dermatitis) as they grow. Typical examples include neonatal acne, seborrheic eczema, contact dermatitis (rash), and atopic dermatitis (AD). Atopic dermatitis is a disease whose main lesion is pruritic eczema with repeated exacerbations and remissions. It is characterized (Non-Patent Document 1). In infancy, it has been reported that it is related to the subsequent allergic march (the development of allergies one after another starting from the onset of one allergy), and prevention of onset, early detection and therapeutic intervention are important. It is said that

[0003] Diaper dermatitis (diaper rash), which frequently occurs in infants, is dermatitis mainly characterized by erythema on the diaper wearing site, and is considered to be a non-allergic temporary irritant contact dermatitis. Such temporary irritant contact dermatitis develops upon contact with an irritating causative agent, but is improved by removing the causative agent. However, if the condition continues without appropriate treatment, it may lead to the development of allergic diseases including atopic dermatitis due to the deterioration of skin barrier function, penetration of allergens, and establishment of sensitization due to long-term exposure to allergens. Therefore, like atopic dermatitis, prevention of its onset, early detection and therapeutic intervention are important (Non-Patent Document 2).

[0004] Diagnosis by a dermatologist is important for understanding the eczema condition, but the decision to see a medical institution is left to the subjective judgment of the guardian, and the value standard differs from individual to individual. It has the potential to be missed. Therefore, a technology that can more easily determine the eczema state is considered to be a technology that brings about appropriate skin care for infants and supports their healthy growth.

[0005] In recent years, techniques have been developed for examining the current and future physiological conditions of human beings by analyzing nucleic acids such as DNA and RNA in biological samples. Biological nucleic acids can be extracted from body fluids such as blood, secretions, tissues, and the like. 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 document]

[0006] [Patent document 1] WO 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 [Outline of the invention] [Problems to be solved by the invention]

[0008] The present invention relates to providing a detection marker for detecting the severity of infant diaper dermatitis and a method for detecting the severity of infant diaper dermatitis using the detection marker. [Means for solving the problem]

[0009] The present inventor collected SSL from the abdomen, waist, left and right inguinal regions, and left and right buttocks of infants with diaper dermatitis, and comprehensively analyzed the expression state of RNA contained in SSL as sequence information. The expression level of the gene was significantly correlated with the severity of diaper dermatitis in infants, and it was found that the severity of diaper dermatitis in infants can be detected using this as an index.

[0010] The present invention relates to the following 1) to 3). 1) For biological samples collected from test infants, including the step of measuring the expression level of at least one gene or its expression product selected from the four gene groups of GALNT3, CMTM6, SLC35E1 and EID3 A method for detecting the severity of diaper dermatitis. 2) detecting the severity of infant diaper dermatitis used in the method of 1), which contains an oligonucleotide that specifically hybridizes with the gene or a nucleic acid derived therefrom, or an antibody that recognizes the expression product of the gene; test kit for 3) A detection marker for detecting the severity of infant diaper dermatitis, comprising at least one gene selected from the 14 gene groups shown in Table 1 or its expression product.

[0011]

table 1

Effect of the invention

[0012] INDUSTRIAL APPLICABILITY According to the present invention, it is possible to easily and noninvasively detect the degree of diaper dermatitis in infants. As a result, parents can grasp the state of diaper dermatitis in infants and toddlers based on the objective index, and can take appropriate measures against the diaper dermatitis. [Brief description of the drawing]

[0013]

Figure 1

[0014] All patents, non-patents, and other publications cited herein are hereby incorporated by reference in their entirety.

[0015] As used herein, the term "nucleic acid" or "polynucleotide" means 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.

[0016] In the present invention, the term "gene" refers to double-stranded DNA containing human genomic DNA, single-stranded DNA (positive strand) containing cDNA, and single-stranded DNA (complementary strand) having a sequence complementary to the positive strand. , and fragments thereof, in which some biological information is contained in the sequence information of the bases that constitute DNA. In addition, the "gene" in the present invention includes not only a "gene" represented by a specific nucleotide sequence, but also its homologues (i.e., homologs or orthologs), mutants such as genetic polymorphisms, and derivatives. be. Here, the names of the genes disclosed in this specification follow the Official Symbols described in NCBI ([www.ncbi.nlm.nih.gov / ]).

[0017] In the present invention, the "expression product" of a gene is a concept that includes transcription products and translation products of a gene. A “transcription product” is an RNA produced by transcription from a gene (DNA), and a “translation product” is a protein encoded by a gene that is translated and synthesized based on the RNA.

[0018] In the present invention, "diaper dermatitis" refers to inflammation that occurs on the skin in contact with a diaper. Clinically, erythema, papules, maceration, and desquamation are seen. As the symptoms worsen, erythema changes to erythema with edema, papules to pustules, and maceration to erosions. Areas where diaper dermatitis is likely to occur include the urination area, anus area, buttocks, groin area, waist area, abdomen area, thigh area, and the vicinity of these areas. "Infants" is broadly defined as "children" before the onset of secondary sex characteristics, specifically a concept that includes children under the age of 12, preferably from 0 years old to school children, specifically Generally refers to infants from 0 to 5 years old.

[0019] The “severity” of infant diaper dermatitis refers to the severity of the symptoms of infant diaper dermatitis, such as no symptoms, mild, mild, mild to moderate, moderate, moderate to severe, and severe. are categorized. The severity of infant diaper dermatitis can be determined based on a known evaluation score for evaluating symptoms of diaper dermatitis (hereinafter also referred to as "diaper dermatitis score"). As the diaper dermatitis score, for example, for each evaluation site, 4 symptoms of erythema, papules, maceration and desquamation are 0: no symptoms, 1: mild, 2: mild, 3: mild to moderate, 4: moderate , 5: Moderate to severe, 6: Severe, with a score of 7 levels, and the total score of all the evaluation sites (Pediatric Dermatology (2014) Vol. 31 No. 1 p. 1-7, and Dermatology (2000) 200 p.238-243). Taking the case of 6 evaluation sites (abdomen, waist, left and right inguinal regions, left and right buttocks) as an example, the severity of diaper dermatitis is based on the sum of evaluation scores, 0: no symptoms, 1 to 24. 25-48: mild, 49-72: mild to moderate, 73-96: moderate, 97-120: moderate to severe, 121-144: severe. In the present invention, the diaper dermatitis score itself may be used as an indicator of the severity of infant diaper dermatitis.

[0020] In the present invention, "detection" of the severity of infant diaper dermatitis can also be rephrased by terms such as inspection, measurement, determination, or evaluation support. The terms "detection", "examination", "measurement", "determination" or "evaluation" of the severity of infant diaper dermatitis in the present invention include diagnosis of the severity of infant diaper dermatitis by a doctor. is not.

[0021] As shown in Examples described later, SSL was collected from the abdomen, waist, left and right inguinal regions, and left and right buttocks of infants with diaper dermatitis, and the data (read count value) of the expression level of the SSL-derived RNA was obtained. 8 genes shown in Table 2, 10 genes shown in Table 3, and 7 genes shown in Table 4 were selected as feature amount genes by the following procedures 1), 2), or 3). It was also shown that the severity of infant diaper dermatitis can be detected by a discriminant (diaper dermatitis score prediction model) using the feature gene. Therefore, a gene or its expression product selected from the gene groups shown in Tables 2 to 4 can be used as a detection marker, and the severity of infant diaper dermatitis can be detected based on the expression level. Furthermore, the genes indicated in bold with * in Tables 2 to 4 are genes that have not been reported to be related to infant diaper dermatitis so far, and genes selected from these gene groups or their expression products. is a novel detectable marker for detecting the severity of infant diaper dermatitis. 1) Convert the read count value to an RPM value corrected for the difference in the total number of reads between sample infants, add an integer 1 to it, and convert it to a base 2 logarithm value ((log 2 (RPM+1) value) and Spearman's rank correlation coefficient for the diaper dermatitis score. A gene (upper gene) with a large Spearman's rank correlation coefficient (ρ) is selected as a feature gene. 2) Convert the read count value to an RPM value corrected for differences in the total number of reads between sample infants, and add the integer 1 to the base 2 logarithm (log 2 Machine learning (machine learning algorithm; random forest) is performed with (RPM + 1) value) as the explanatory variable and the diaper dermatitis score as the objective variable, and the genes with the highest variable importance based on the Gini coefficient calculated by the random forest function is selected as a feature gene. 3) Logarithmic value (log 2 Machine learning (machine learning algorithm; BORUTA method) is performed with the (RPM + 1) value) as the explanatory variable and the diaper dermatitis score as the objective variable. Select as

[0022] [Table 2]

[0023] [Table 3]

[0024] [Table 4]

[0025] Among the genes shown in Tables 2 to 4, the four types of genes, GALNT3, CMTM6, SLC35E1 and EID3, are the gene groups shown in Table 2, the gene groups shown in Table 3, and the gene groups shown in Table 4. In, any two or more gene clusters are duplicated genes. In addition, among these, GALNT3 and CMTM6 are overlapping genes in all of these three gene clusters. The four genes, GALNT3, CMTM6, SLC35E1 and EID3, are genes that have not been reported to be associated with infant diaper dermatitis (marked with * and shown in bold in each table). As shown in Examples described later, the severity of infant diaper dermatitis is detected by a discriminant (diaper dermatitis score prediction model) using at least one or more genes selected from these genes as feature amount genes. was shown to be possible. Therefore, at least one selected from genes selected from the four gene groups of GALNT3, CMTM6, SLC35E1 and EID3 or their expression products is used as a detection marker, and based on its expression level, symptoms of infant diaper dermatitis are detected. degree can be detected. Further, one or more expression levels selected from the genes selected from the four gene groups or their expression products, and the 14 gene groups shown in Table 5 (GALNT3 from the genes shown in Tables 2 to 4) , CMTM6, SLC35E1 and EID3 genes), and the expression level of one or more selected from genes or their expression products, based on the severity of diaper dermatitis in the test infant detection is possible. By detecting the severity of diaper dermatitis in infants, the state of diaper dermatitis in infants, for example, the presence or absence of onset of dermatitis, the degree of progression of dermatitis, the degree of healing of dermatitis, therapeutic or preventive effect on dermatitis, etc. can be grasped.

[0026] [Table 5]

[0027] Each of the four genes, GALNT3, CMTM6, SLC35E1 and EID3, can be independently used as a detection marker for detecting the severity of infant diaper dermatitis. A combination of 3 or more, more preferably 4, is used. Among them, it is preferable to select two or more types including GALNT3 and CMTM6. In addition, at least one or more, preferably two or more, and more preferably three genes selected from Table 5 to be combined with the four genes of GALNT3, CMTM6, SLC35E1, and EID3 That's it. Furthermore, at least one or more, preferably two or more, and more preferably three or more genes are preferably selected from the genes indicated in bold with * in the table that have not been reported to be related to infant diaper dermatitis. In the present invention, it is preferable to select combinations of genes listed in Table 2, Table 3 or Table 4 above.

[0028] The gene that can be a detection marker for detecting the severity of infant diaper dermatitis (hereinafter also referred to as "target gene") includes, as long as it can be a biomarker for detecting the severity of infant diaper dermatitis, A gene having a base sequence substantially identical to the base sequence of the DNA constituting the gene is also included. Here, the substantially identical base sequence is, for example, using the homology calculation algorithm NCBI BLAST, with the following conditions: expected value = 10; gaps allowed; filtering = ON; match score = 1; mismatch score = -3 It means that it has 90% or more, preferably 95% or more, more preferably 98% or more, and still more preferably 99% or more identity with the base sequence of the DNA constituting the gene when searched with .

[0029] In the method for detecting the severity of infant diaper dermatitis of the present invention, target genes are selected from four gene groups, GALNT3, CMTM6, SLC35E1 and EID3, in one embodiment, for biological samples collected from test infants. Measuring the level of expression of at least one gene or its expression product. In another embodiment, the expression level of at least one gene or its expression product selected from the four gene groups and the expression level of the expression product thereof selected from the four gene groups and the 14 gene groups shown in Table 5 are selected from the biological sample collected from the test infant. and measuring the expression level of at least one gene or expression product thereof.

[0030] Infants to be tested in the present invention include, for example, infants who desire or need detection of diaper dermatitis. For example, the infants to be tested are infants who have developed diaper dermatitis, infants suspected of developing diaper dermatitis, infants genetically predisposed to diaper dermatitis, or close relatives such as brothers and sisters who have diaper dermatitis. Infants who have or have had symptoms are included.

[0031] The biological sample used in the present invention may be any cell, tissue, or biomaterial in which the expression of the gene of the present invention changes. Specific examples include organs, skin, blood, urine, saliva, sweat, stratum corneum, superficial skin lipids (SSL), body fluids such as tissue exudates, serum prepared from blood, blood plasma, feces, hair, etc. preferably skin or superficial skin lipids (SSL), more preferably superficial skin lipids (SSL). The site of the skin from which SSL is collected is not particularly limited, and includes any site of the body such as the head, face, neck, trunk, limbs, etc., preferably the site where infant diaper dermatitis is likely to occur. , for example, the urinary part, the anal part, the buttocks, the groin, the waist, the abdomen, the thigh, and the vicinity thereof. The site of the skin from which SSL is collected may be either a rash where infant diaper dermatitis has developed or a non-rash site where infant diaper dermatitis has not developed, but preferably the rash or the vicinity of the rash. is the non-erupted part of the Here, the vicinity of the rash refers to a range within 10 cm adjacent to the rash.

[0032] Here, "superficial skin lipid (SSL)" refers to a fat-soluble fraction present on the surface of the skin, and is sometimes called sebum. In general, SSL mainly contains secretions secreted from exocrine glands such as sebaceous glands in the skin, and is present on the skin surface in the form of a thin layer covering the skin surface. SSL contains RNA expressed in skin cells. (See Patent Document 1 above). In the present invention, unless otherwise specified, "skin" is a general term for areas including stratum corneum, epidermis, dermis, hair follicles, and tissues such as sweat glands, sebaceous glands and other glands.

[0033] Any means used to collect or remove SSL from the skin can be used to collect SSL from the skin of the infant being tested. Preferably, an SSL absorbent material, an SSL adhesive material, or an instrument that scrapes the SSL off the skin, as described below, can be used. The SSL absorbent material or SSL adhesive material is not particularly limited as long as it has affinity for SSL, and examples thereof include polypropylene and pulp. More detailed examples of procedures for collecting SSL from the skin include a method of absorbing SSL into sheet-like materials such as blotting paper and blotting film, a method of adhering SSL to a glass plate, tape, etc., a spatula, a scraper, etc. and a method of scraping off and recovering the SSL. In order to improve the absorbability of SSL, an SSL absorbent material pre-impregnated with a highly fat-soluble solvent may be used. On the other hand, if the SSL absorbent material contains a highly water-soluble solvent or water, the adsorption of SSL is inhibited. The SSL absorbent material is preferably used dry.

[0034] RNA-containing SSL collected from a test infant may be stored for a period of time. The collected SSL is preferably stored under low-temperature conditions as soon as possible after collection in order to minimize degradation of the contained RNA. The temperature condition for storing the RNA-containing SSL in the present invention may be 0°C or lower, preferably -20±20°C to -80±20°C, more preferably -20±10°C to -80±10°C. , More preferably -20 ± 20°C to -40 ± 20°C, more preferably -20 ± 10°C to -40 ± 10°C, still more preferably -20 ± 10°C, still more preferably -20 ± 5°C . The storage period of the RNA-containing SSL under the low-temperature conditions is not particularly limited, but is preferably 12 months or less, such as 6 hours or more and 12 months or less, more preferably 6 months or less, such as 1 day or more and 6 months or less, More preferably 3 months or less, for example 3 days or more and 3 months or less.

[0035] In the present invention, targets for measuring the expression level of the target gene or its expression product include cDNA artificially synthesized from RNA, DNA encoding the RNA, protein encoded by the RNA, and interaction with the protein. molecules that interact with the RNA, molecules that interact with the DNA, and the like. Here, molecules that interact with RNA, DNA or protein include DNA, RNA, protein, polysaccharides, oligosaccharides, monosaccharides, lipids, fatty acids, phosphorylated compounds, alkylated products, sugar adducts thereof, and the like, and Any one of the above complexes may be mentioned. In addition, the expression level comprehensively means the expression level and activity of the gene or expression product.

[0036] In the method of the present invention, as a preferred embodiment, SSL is used as a biological sample. In this case, the expression level of RNA contained in SSL is analyzed, specifically after converting RNA into cDNA by reverse transcription. , the cDNA or its amplification product is measured. For extraction of RNA from SSL, methods commonly used for extraction or purification of RNA from biological samples, such as the phenol / chloroform method, the AGPC (acid guanidinium thiocyanate-phenol-chloroform extraction) method, or TRIzol® ), RNeasy (registered trademark), methods using columns such as QIAzol (registered trademark), methods using special magnetic particles coated with silica, methods using Solid Phase Reversible Immobilization magnetic particles, commercially available such as ISOGEN Extraction with an RNA extraction reagent or the like can be used.

[0037] A primer targeting a specific RNA to be analyzed may be used for the reverse transcription, but random primers are preferably used for more comprehensive nucleic acid storage and analysis. A common reverse transcriptase or reverse transcription reagent kit can be used for the reverse transcription. Preferably, a highly accurate and efficient reverse transcriptase or reverse transcription reagent kit is used, examples of which include M-MLV Reverse Transcriptase and variants thereof, or commercially available reverse transcriptase or reverse transcription reagent kit, Examples include PrimeScript (registered trademark) Reverse Transcriptase series (Takara Bio Inc.) and SuperScript (registered trademark) Reverse Transcriptase series (Thermo Scientific). SuperScript (registered trademark) III Reverse Transcriptase, SuperScript (registered trademark) VILO cDNA Synthesis kit (both from Thermo Scientific) and the like are preferably used. In the elongation reaction in the reverse transcription, the temperature is adjusted to preferably 42°C ± 1°C, more preferably 42°C ± 0.5°C, and even more preferably 42°C ± 0.25°C, while the reaction time is preferably 60 minutes or longer. More preferably, it is adjusted to 80 to 120 minutes.

[0038] Typical methods for measuring expression levels include PCR, real-time RT-PCR, multiplex PCR, SmartAmp, LAMP, etc., using DNA that hybridizes to RNA, cDNA, or DNA as primers. hybridization methods using nucleic acids that hybridize to these as probes (DNA chips, DNA microarrays, dot blot hybridizations, slot blot hybridizations, northern blot hybridizations, etc.), methods for determining base sequences ( sequencing), or a combination of these.

[0039] In PCR, a primer pair targeting a specific DNA to be analyzed may be used to amplify only one specific DNA, but a plurality of primer pairs may be used to amplify a plurality of specific DNAs at the same time. good too. Preferably, said PCR is a multiplex PCR. Multiplex PCR is a method of simultaneously amplifying multiple gene regions by simultaneously using multiple primer pairs in a PCR reaction system. Multiplex PCR can be performed using a commercially available kit (eg, Ion AmpliSeq Transcriptome Human Gene Expression Kit; Life Technologies Japan Co., Ltd., etc.). The temperature of the annealing and extension reaction in the PCR depends on the primers used, so it cannot be generalized. °C, more preferably 62°C ± 0.25°C. Therefore, in the PCR, annealing and extension reactions are preferably performed in one step. The time for the annealing and extension reaction steps can be adjusted depending on the size of the DNA to be amplified and the like, but is preferably 14 to 18 minutes. The denaturation reaction conditions in the PCR can be adjusted depending on the DNA to be amplified, but are preferably 95-99° C. for 10-60 seconds. Reverse transcription and PCR at the temperatures and times described above can be performed using a thermal cycler commonly used for PCR.

[0040] Purification of the reaction product obtained by the PCR is preferably performed by size separation of the reaction product. Size separation allows separation of the desired PCR reaction product from primers and other impurities contained in the PCR reaction. Size separation of DNA can be performed by, for example, a size separation column, a size separation chip, magnetic beads that can be used for size separation, or the like. Preferred examples of magnetic beads that can be used for size separation include Solid Phase Reversible Immobilization (SPRI) magnetic beads such as Ampure XP.

[0041] Purified PCR reaction products may be subjected to further processing as required for subsequent quantitative analysis. For example, for DNA sequencing, the purified PCR reaction product is prepared in an appropriate buffer solution, the PCR primer region contained in the PCR-amplified DNA is cleaved, and the amplified DNA is added with an adapter sequence. may be added. For example, a purified PCR reaction product is prepared in a buffer solution, PCR primer sequences are removed from the amplified DNA and adapter ligation is performed, and the resulting reaction product is amplified as necessary for quantitative analysis. of libraries can be prepared. These operations are performed, for example, using 5×VILO RT Reaction Mix attached to SuperScript (registered trademark) VILO cDNA Synthesis kit (Life Technologies Japan Co., Ltd.) and Ion AmpliSeq Transcriptome Human Gene Expression Kit (Life Technologies Japan Co., Ltd.). 5×Ion AmpliSeq HiFi Mix and Ion AmpliSeq Transcriptome Human Gene Expression Core Panel attached to the kit can be used according to the protocol attached to each kit.

[0042] When measuring the expression level of a target gene or a nucleic acid derived from it using the Northern blot hybridization method, for example, the probe DNA is first labeled with a radioactive isotope, a fluorescent substance, etc., and then the resulting labeled DNA is , and hybridize with biological sample-derived RNA transferred to a nylon membrane or the like according to a conventional method. Then, there is a method of measuring the formed double strand of labeled DNA and RNA by detecting a signal derived from the label.

[0043] When the expression level of a target gene or a nucleic acid derived therefrom is measured using the RT-PCR method, for example, first, cDNA is prepared from RNA derived from a biological sample according to a conventional method, and the target gene of the present invention is obtained using this as a template. A pair of primers prepared for amplification (the positive strand that binds to the above cDNA (− strand) and the reverse strand that binds to the + strand) is hybridized with this. Thereafter, 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 for detecting the labeled double-stranded DNA produced by performing the above-mentioned PCR using primers that have been labeled with RI, a fluorescent substance, etc. in advance, etc. can be done.

[0044] When measuring the expression level of a target gene or a 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. , mRNA expression levels can be measured by binding labeled cDNA or cRNA prepared from mRNA onto a microarray and detecting the label on the microarray. The nucleic acids immobilized on the array may be nucleic acids that hybridize specifically (that is, substantially only to the target nucleic acid) under stringent conditions. It may be a nucleic acid having a sequence or a nucleic acid consisting of a partial sequence. Here, the “partial sequence” includes nucleic acids consisting of at least 15 to 25 bases. Here, stringent conditions are usually washing conditions of "1 x SSC, 0.1% SDS, 37°C", and more stringent hybridization conditions are "0.5 x SSC, 0.1% SDS, 42°C". A more stringent hybridization condition is "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) and others.

[0045] When the expression level of a target gene or a nucleic acid derived therefrom is measured by sequencing, for example, analysis using a next-generation sequencer (eg, Ion S5 / XL system, Life Technologies Japan Co., Ltd.) can be used. Based on the number of reads generated by sequencing (read count), RNA expression can be quantified.

[0046] Probes or primers used for the above measurements, that is, primers for specifically recognizing and amplifying the target gene of the present invention or nucleic acids derived therefrom, or for specifically detecting the RNA or nucleic acids derived therefrom Probes fall into this category, and they can be designed based on the nucleotide sequence that constitutes the target gene. Here, "specifically recognize" means that, for example, in Northern blotting, substantially only the target gene of the present invention or a nucleic acid derived from it can be detected, and for example, in RT-PCR, substantially only the nucleic acid is amplified, it means that the detected product or product can be determined to be the gene or the nucleic acid derived therefrom. Specifically, an oligonucleotide containing a certain number of nucleotides complementary to a DNA consisting of a nucleotide sequence constituting the target gene of the present invention or its complementary strand can be used. As used herein, the term "complementary strand" refers to one strand of a double-stranded DNA consisting of base pairs of A:T (U in the case of RNA) and G:C against the other strand. In addition, "complementary" is not limited to the case of a completely complementary sequence in the certain number of consecutive nucleotide regions, preferably 80% or more, more preferably 90% or more, further preferably 95% or more, more preferably 80% or more, more preferably 90% or more. More preferably, they should have 98% or more nucleotide sequence identity. The identity of base sequences can be determined by algorithms such as BLAST. When such oligonucleotides are used as primers, they only need to be capable of specific annealing and chain extension. Those having a chain length of preferably 50 bases or less, more preferably 35 bases or less are included. In addition, when used as a probe, it only needs to be capable of specific hybridization, and has at least a partial or entire sequence of DNA (or its complementary strand) consisting of the base sequence that constitutes the target gene of the present invention, such as 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, more preferably 25 bases or less is used. Here, "oligonucleotide" can be DNA or RNA, and may be synthetic or natural. Also, the probes used for hybridization are usually labeled ones.

[0047] In addition, protein chip analysis, immunoassay ( ELISA, etc.), 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)) can be used, and can be appropriately selected depending on the subject. For example, when a protein is used as an object to be measured, an antibody that specifically recognizes the expression product of the present invention, specifically a structural characteristic site ( It is carried out by contacting an antibody that recognizes the epitope) with a biological sample, detecting the polypeptide or protein in the sample that binds to the antibody, and measuring the level. For example, according to Western blotting, after using the above antibody as a primary antibody, an antibody that binds to the primary antibody labeled with a radioisotope, fluorescent substance, enzyme, etc. is used as a secondary antibody, and the primary antibody is Labeling is performed, and signals derived from these labeling substances are measured with a radiometer, a fluorescence detector, or the like. The antibody against the translation product may be either a polyclonal antibody or a monoclonal antibody. These antibodies can be produced according to known methods. Specifically, a polyclonal antibody is obtained by immunizing a non-human animal such as a rabbit using a protein expressed in Escherichia coli or the like and purified according to a conventional method, or by synthesizing a partial polypeptide of the protein according to a conventional method, It can be obtained from the serum of the immunized animal according to a conventional method. On the other hand, monoclonal antibodies are obtained by immunizing a non-human animal such as a mouse with a protein expressed in Escherichia coli or the like and purified according to a conventional method or a partial polypeptide of the protein, and fusing the obtained spleen cells with myeloma cells. It can be obtained from prepared hybridoma cells. Monoclonal antibodies may also be generated using phage display (Griffiths, A.D.; Duncan, A.R., Current Opinion in Biotechnology, Volume 9, Number 1, February 1998, pp. 102-108(7)).

[0048] Thus, the expression level of the target gene of the present invention or its expression product in a biological sample collected from a test infant is measured, and the severity of diaper dermatitis in the test infant is detected based on the expression level. . In addition, in the present invention, the expression level of the target gene of the present invention or its expression product in a biological sample collected from the test infant is measured at least at two times, and the change in the expression level or the amount of change is used as an index, and the test infant It is possible to detect the presence or absence of change in the degree of diaper dermatitis or the degree of change. Specifically, detection is performed by comparing the measured expression level of the target gene of the present invention or its expression product with a preset cutoff value (reference value). When analyzing the expression levels of multiple target genes by sequencing, as described above, the read count value, which is the expression level data, and the RPM value obtained by correcting the difference in the total read number between samples , the value obtained by converting the RPM value to base 2 logarithm (log 2 RPM value) or base 2 logarithm with integer 1 added (log 2 (RPM + 1) value), or normalized count value using DESeq2 (Love MI et al. Genome Biol. 2014) or base 2 logarithm value (log 2 (Normalized count+1) value) is preferably used as an index. In addition, it is calculated by fragments per kilobase of exon per million reads mapped (FPKM), reads per kilobase of exon per million reads mapped (RPKM), transcripts per million (TPM), etc., which are commonly used as quantitative values ​​for RNA-seq. can be a value. Alternatively, it may be a signal value obtained by a microarray method and its correction value. In addition, when analyzing only a specific target gene by RT-PCR, etc., a method of converting the expression level of the target gene into a relative expression level based on the expression level of the housekeeping gene and analyzing it, or A method of quantifying the absolute copy number using a plasmid containing the region of the target gene (absolute quantification) and analyzing is preferred. It may be the copy number obtained by the digital PCR method.

[0049] Here, the "cutoff value" ("reference value") can be determined in advance based on the relationship between the diaper dermatitis score and the expression level of the target gene of the present invention or its expression product. For example, a population is divided into diaper dermatitis status, i.e. no symptoms, mild, mild, mild-to-moderate, moderate, moderate-to-severe, severe groups according to the diaper dermatitis score, and the target in each group A value determined with reference to statistical values ​​such as the average value and standard deviation of the expression level of the gene or its expression product can be determined as a cutoff value (reference value) for determining belonging to each group. When multiple types of genes are used as target genes, it is preferable to determine a cutoff value (reference value) for each gene or its expression product. Groups may be formed according to sex, race, and age.

[0050] Furthermore, a discriminant (prediction model) is constructed using the measured value of the expression level of the target gene of the present invention or its expression product, and the degree of diaper dermatitis is detected using the discriminant. can be done. For example, by machine learning using the measurement value of the expression level of the target gene or its expression product as an explanatory variable and the diaper dermatitis score (for example, the value obtained by converting the score into a deviation value) as the objective variable, the symptoms of diaper dermatitis An optimal discriminant (prediction model) can be constructed to detect the degree. Then, the expression level of the target gene of the present invention or its expression product is similarly measured from the biological sample collected from the test infant, the obtained measured value is input to the discriminant (prediction model), and the discriminant is The obtained result (predicted value of diaper dermatitis score) can be detected as the degree of diaper dermatitis in the test infant.

[0051] Furthermore, by using the expression level of the target gene or its expression product derived from diaper dermatitis infants with different severity and the expression level of the target gene or its expression product derived from healthy infants, diaper skin with different severity Discriminant formula to separate infants with inflammation (e.g., 2 or more groups selected from mild, mild, mild-to-moderate, moderate, moderate-to-severe, severe, etc.) and healthy infants (no symptoms) A (prediction model) can be constructed and the discriminant can be used to detect the severity of infant diaper dermatitis. That is, the measured values ​​of the expression levels of target genes or their expression products derived from a group of diaper dermatitis infants with different degrees of diaper dermatitis and the measured values ​​of the expression levels of target genes or their expression products derived from healthy infants were used as teaching samples, and diapers with different degrees of severity were used. Discriminant formula to divide infants with dermatitis (e.g., 2 or more groups selected from mild, mild, mild-to-moderate, moderate, moderate-to-severe, severe, etc.) and healthy infants (no symptoms) A (prediction model) is constructed, and a cutoff value (reference value) for discriminating diaper dermatitis infants with different degrees of severity is obtained based on the discriminant. Then, similarly measure the expression level of the target gene or its expression product from the biological sample collected from the test infant, substitute the obtained measured value into the discriminant, and cut off the result obtained from the discriminant By comparing with the value (reference value), the degree of diaper dermatitis in the subject infant can be detected.

[0052] As an algorithm for constructing the discriminant (prediction model), a known one such as an algorithm used for machine learning can be used. Examples of machine learning algorithms include Random forest, Support Vector Machine with linear kernels (SVM linear), Support Vector Machine with rbf kernels (SVM rbf), Neural nets, Generalized linear models. model), regularized linear discriminant analysis, regularized logistic regression, Lasso (Least Absolute Shrinkage and Selection Operator) regression, and the like. Enter verification data into the constructed prediction model, calculate the prediction value, and select the model that best matches the prediction value with the actual measurement value, for example, the model with the highest accuracy rate (Accuracy) as the optimal prediction model. can be done. In addition, the detection rate (Recall), precision (Precision), and their harmonic average F value can be calculated from the predicted value and the actual measured value, and the model with the largest F value can be selected as the optimal prediction model. . In addition, the root mean square error (RMSE) between the predicted value and the measured value is used as an accuracy evaluation index of the prediction model, and the model with the smallest RMSE can be selected as the optimum prediction model.

[0053] A method for determining the cutoff value (reference value) is not particularly limited, and can be determined according to a known method. For example, it can be obtained from an ROC (Receiver Operating Characteristic Curve) curve created using a discriminant (prediction model). In the ROC curve, the vertical axis is the probability of positive results in positive subjects (sensitivity), and the horizontal axis is the value obtained by subtracting the probability of negative results in negative subjects (specificity) from 1 (false positive rate). be done. Regarding "true positive (sensitivity)" and "false positive (1-specificity)" shown in the ROC curve, the value (Youden index) can be used as a cutoff value (reference value).

[0054] A test kit for detecting the severity of infant diaper dermatitis of the present invention contains a test reagent for measuring the expression level of the target gene of the present invention or its expression product in a biological sample isolated from a test infant. It is. Specifically, reagents for nucleic acid amplification and hybridization, which contain oligonucleotides (for example, primers for PCR) that specifically bind (hybridize) with the target gene of the present invention or nucleic acids derived therefrom, or Reagents for immunoassays containing antibodies that recognize the expression product (protein) of the target gene of the present invention, and the like. Oligonucleotides, antibodies and the like included in the kit can be obtained by known methods as described above. In addition to the above antibodies and nucleic acids, the test kit also contains labeling reagents, buffers, chromogenic substrates, secondary antibodies, blocking agents, tools necessary for testing, control reagents used as positive and negative controls, Equipment for collecting biological samples (eg, blotting film for collecting SSL, etc.) and the like can be included.

[0055] The present invention further discloses the following aspects regarding the above-described embodiments.

[0056] <1> For biological samples collected from test infants, including the step of measuring the expression level of at least one gene or its expression product selected from the four gene groups of GALNT3, CMTM6, SLC35E1 and EID3 method for detecting the severity of diaper dermatitis in children.

[0057] <2> Preferably 2 or more, more preferably 3 or more, more preferably 2 or more, more preferably 4, including GALNT3 and CMTM6 selected from the 4 gene groups of GALNT3, CMTM6, SLC35E1 and EID3 The detection method according to <1>, wherein expression levels of genes or expression products thereof are measured for all species. <3> Preferably, the detection method according to <1> or <2>, further comprising the step of measuring the expression level of at least one gene or its expression product selected from the 14 gene groups shown in Table 5 above. . <4> Preferably 2 or more, more preferably 3 or more, more preferably 3 or more selected from the 14 gene groups shown in Table 5, and more preferably with infant diaper dermatitis indicated in bold with * in Table 5 The detection method according to <3>, wherein the expression level of at least one, preferably two or more, more preferably three or more genes or their expression products selected from unreported genes is measured. <5> Preferably, the detection method according to any one of <1> to <4>, wherein the expression level of the gene or expression product thereof shown in Table 2, Table 3 or Table 4 is measured. <6> The biological sample is preferably an organ, skin, blood, urine, saliva, sweat, stratum corneum, superficial skin lipid (SSL), body fluid such as tissue exudate, serum prepared from blood, plasma , stool or hair, more preferably skin or superficial skin lipids (SSL), still more preferably superficial skin lipids (SSL), <1> to <5>. <7> The object to be measured for the expression level of the gene or its expression product is preferably cDNA artificially synthesized from RNA, DNA encoding the RNA, a protein encoded by the RNA, or interacting with the protein. The detection method according to any one of <1> to <6>, wherein the molecule interacts with the RNA, the molecule interacts with the RNA, or the molecule interacts with the DNA. <8> The detection according to any one of <1> to <5>, wherein the expression level of the gene or its expression product is preferably the expression level of mRNA collected from superficial skin lipids (SSL) of the test infant. Method. <9> The test infant is an infant who has developed infant diaper dermatitis, an infant who is suspected of developing infant diaper dermatitis, an infant who is genetically predisposed to infant diaper dermatitis, or a relative such as siblings The detection method according to <8>, wherein the infant is developing or has developed infant diaper dermatitis. <10> The detection method according to <8> or <9>, wherein the skin of the subject infant is preferably the skin of the urinary region, anus, buttocks, groin, waist, abdomen, or thigh. <11> The skin site from which superficial skin lipids (SSL) are collected may be either the rash area where infant diaper dermatitis develops or the non-rash area where infant diaper dermatitis does not develop. , The detection method according to any one of <8> to <10>, which is preferably an erupted area or an erupted area near the erupted area. <12> The detection method according to any one of <1> to <11>, comprising detecting the severity of diaper dermatitis in the test infant based on the expression level of the gene or its expression product. <13> Preferably, detecting the severity of infant diaper dermatitis using a discriminant (prediction model) based on the expression level of the gene or its expression product, Any of <1> to <12>, wherein the discriminant (prediction model) is constructed by machine learning with the measured value of the expression level of the gene or its expression product as an explanatory variable and the diaper dermatitis score as an objective variable. The detection method according to . <14> The severity of infant diaper dermatitis is 0: no symptoms, 1: mild, 2: mild, 3: mild to moderate, for each evaluation site, for 4 symptoms of erythema, papules, maceration and desquamation. 4: Moderate, 5: Moderate to severe, 6: Severe 7-level scores are added, and the sum of the scores of all evaluation sites (total value) corresponds to the degree of severity <1> to <13. The detection method according to any of >. <15> The detection method according to <14>, wherein the evaluation sites are the abdomen, waist, left and right inguinal regions, and left and right buttocks of the infant. <16> Infant diaper skin used in the detection method of <1> to <15>, containing 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 A test kit for detecting the severity of inflammation. <17> A detection marker for detecting the severity of infant diaper dermatitis, comprising at least one gene or its expression product selected from the gene group indicated in bold with * in Tables 2 to 4 above. <18> A detection marker for detecting the severity of infant diaper dermatitis according to <17>, which is at least one gene selected from the four gene groups of GALNT3, CMTM6, SLC35E1 and EID3 or an expression product thereof. . <19> Infant diaper dermatitis of at least one gene or its expression product selected from the group of genes indicated in bold with * in Tables 2 to 4 above derived from biological samples collected from test infants Use as a marker for detection of disease severity. <20> Use as a detection marker according to <19>, which is at least one gene or its expression product selected from the four gene groups of GALNT3, CMTM6, SLC35E1 and EID3. <21> The use according to <19> or <20>, wherein the gene or its expression product is mRNA contained in the lipids on the skin surface of the test infant.

Example

[0058] Example 1 Detection of diaper dermatitis severity using RNA extracted from SSL 1) Measurement of test infants and diaper dermatitis score The subjects were 59 Japanese male and female infants aged 3 to 8 months. A dermatologist scored the 4 symptoms of erythema, papules, maceration and desquamation based on the criteria shown in Table 6 by examining the abdomen, waist, left and right inguinal regions, and left and right buttocks of the infants to be tested. A value (total value) obtained by summing up the scores of a total of 6 sites was defined as the diaper dermatitis score. As a result, 0 patients had no symptoms (score 0), 45 patients had mild symptoms (scores 1-24), 13 patients had mild symptoms (scores 25-48), 1 patient had mild to moderate symptoms (scores 49-72), and 1 patient had moderate symptoms. None had severe (score 73-96), 0 had moderate to severe (score 97-120), and 0 had severe (score 121-144).

[0059] [Table 6]

[0060] 2) SSL extraction After collecting sebum from the abdomen, waist, left and right inguinal regions, and left and right buttocks of each test infant using an oil blotting film (5 x 8 cm, made of polypropylene, 3M), the oil blotting film was transferred to a vial and used for RNA extraction. Stored at -80°C until

[0061] 3) RNA preparation and sequencing The blotting film of 2) above was cut into an appropriate size, and RNA was extracted using QIAzol Lysis Reagent (Qiagen) according to the attached protocol. Based on the extracted RNA, reverse transcription was performed at 42° C. for 90 minutes using SuperScript VILO cDNA Synthesis kit (Life Technologies Japan Co., Ltd.) to synthesize cDNA. Random primers attached to the kit were used as primers for the reverse transcription reaction. A library containing DNA derived from the 20802 gene was prepared from the resulting cDNA by multiplex PCR. Multiplex PCR was performed using Ion AmpliSeq Transcriptome Human Gene Expression Kit (Life Technologies Japan Co., Ltd.) [99°C, 2 minutes → (99°C, 15 seconds → 62°C, 16 minutes) × 20 cycles → 4°C, Hold ] conditions. After purifying the obtained PCR product with Ampure XP (Beckman Coulter, Inc.), buffer reconstitution, primer sequence digestion, adapter ligation and purification, and amplification were performed to prepare a library. The prepared library was loaded onto an Ion 540 Chip and sequenced using an Ion S5 / XL system (Life Technologies Japan Co., Ltd.).

[0062] 4) Data analysis (1) Usage data The expression level data (read count value) of the SSL-derived RNA measured in 3) above was obtained and converted into an RPM value corrected for the difference in the total read count between sample infants tested. In selecting feature genes and building a machine learning model, in order to approximate RPM values ​​that follow a negative binomial distribution to a normal distribution, the base-2 logarithm value (log 2 (RPM+1) value) was used. Only 5809 genes for which non-missing expression level data were obtained in 90% or more of the sample tested infants among the expression level data for all sample tested infants were used for the following analysis.

[0063] (2) Dataset division Of the data set of 59 test infants, RNA profile data for 48 children was used as Train data for model construction, and RNA profile data for the remaining 11 children was used as Test data for model accuracy evaluation. Data partitioning was performed using the createDataPartition function of [R] so that the distribution of the objective variable was uniform between the Train data and the Test data.

[0064] (3) Selection of feature gene Using the train data divided in (2), the feature amount genes presumed to be suitable for the classification prediction model were extracted using the following three methods 1) to 3). 1) Extraction based on Spearman's correlation coefficient Spearman's rank correlation coefficient was calculated for the combination of the diaper dermatitis scores of 48 test infants, from which the Train data were derived, and all gene RNA expression data. As a result, the top 8 genes with a large Spearman's rank correlation coefficient (ρ) shown in Table 7 were selected as feature amount genes. The 6 genes indicated in bold with * in the table have not been reported to be related to infant diaper dermatitis.

[0065] [Table 7]

[0066] 2) Extraction based on variable importance Using the caret package of [R], we calculated the importance of each gene based on the Gini coefficient calculated by the random forest function, and selected the top 10 genes that contributed significantly to model creation shown in Table 8. It was selected as a feature gene. The 9 genes marked with * in the table and shown in bold letters have not been reported to be related to infant diaper dermatitis.

[0067] [Table 8]

[0068] 3) Extraction of feature value genes by BORUTA I used a program based on the BORUTA package in [R]. RPM corrected read count value, log 2 A value of (RPM+1) was used. Using the diaper dermatitis score as a correct label, the 7 genes shown in Table 9 that were judged to be "tentative" or "confirmed" were selected as feature amount genes. The 5 genes indicated in bold with * in the table have not been reported to be related to infant diaper dermatitis.

[0069] [Table 9]

[0070] (4) Prediction model for diaper dermatitis score using caret package Top 8 genes highly correlated with diaper dermatitis score, 10 genes with high variable importance by random forest, and 7 genes selected by BORUTA were selected as feature values, and a diaper dermatitis score prediction model was constructed with the caret package. did. Expression level data (log 2 (RPM+1) value) was used as an explanatory variable, and a value obtained by converting the diaper dermatitis score into a deviation value was used as an objective variable. Using the features selected above, train data, linear regression model (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) six algorithms were used to construct a diaper dermatitis score prediction model. To reduce the impact of data bias on the model, model building was cross-validated 10 times. As an index of the best prediction model, RMSE (root mean square error) was calculated, and the model with the smallest value was selected as the best model. Next, for the best model, the feature gene expression level (log 2 (RPM+1) value) was entered to calculate the predicted value of the diaper dermatitis score (Predicted Score). The PEARSON correlation coefficient between the obtained predicted value (prediction score) and the actual diagnostic score was calculated, and the closer this value was to 1, the higher the prediction accuracy of the model.

[0071] (5) Results As shown in Fig. 1, when the eight genes with the highest correlation coefficients with the diaper dermatitis score were used as the feature values, the random forest model (model 1) had the highest accuracy with RMSE = 5.90. The correlation coefficient between the predicted score and diagnostic score based on Test data at that time was 0.902 (p<0.01). When 10 genes with high variable importance by random forest are used as features, the model constructed by SVMrbf (Model 2) has the highest accuracy with RMSE = 6.32, and the correlation coefficient between the prediction score and the diagnosis score by Test data is 0.706 (p=0.015). When the 9 genes extracted by BORUTA are used as features, the model constructed by random forest (model 3) has the highest accuracy with RMSE = 6.39, and the correlation coefficient between the prediction score and the diagnosis score based on the test data at that time is 0.754. (p<0.01). From this, it was clarified that the gene used in the model is an important gene for regression prediction of the infant diaper dermatitis score, and is useful for detecting the severity of infant diaper dermatitis. Especially important genes among multiple feature genes are GALNT3, CMTM6, SLC35E1, and EID3, which are redundantly extracted by two or more of the three methods of extracting feature genes. When a diaper dermatitis score prediction model is constructed using the four types, the model constructed by the neural network has the highest accuracy with RMSE = 6.445, and the correlation coefficient between the prediction score and the diagnosis score based on the test data at that time is 0.698 (p<0.05). In addition, when a diaper dermatitis score prediction model is constructed using two types of GALNT3 and CMTM6 selected from four types or two types of SLC35E1 and EID3, the correlation coefficient between the prediction score and the diagnosis score by Test data is 0.623. (support vector machine of rbf kernel, RMSE=7.023) (p<0.05), 0.614 (neural network, RMSE=7.072) (p<0.05), confirming that it is an important gene. Furthermore, when a diaper dermatitis score prediction model was constructed using only one of GALNT3 or EID3, the correlation coefficient between the prediction score and diagnosis score by Test data was 0.705 (support vector machine of rbf kernel, RMSE=6.724) (p<0.05), 0.649 (linear regression model, RMSE=7.201) (p<0.05), confirming that it is a particularly important gene.

Claims

1. A method for detecting the severity of diaper dermatitis 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 four genes, GALNT3, CMTM6, SLC35E1 and EID3, for skin surface lipids collected from the skin of the test infant.

2. The method according to claim 1, further comprising the step of measuring the expression level of at least one gene selected from the group of 14 genes shown in Table 1 or its expression product. 【Table 1】

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 the skin of the urination area, anus, buttocks, groin, lower back, abdomen or thigh.

5. The method according to claim 1, further comprising detecting the severity of diaper dermatitis in the test infant based on the expression level of the gene or its expression product.

6. A test kit for detecting the severity of infant diaper dermatitis, which is used in the detection method 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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